Immunogenic composition

ABSTRACT

The present application discloses an immunogenic composition comprising at least 2 different N. meningitidis capsular saccharides, wherein one or more is/are selected from a first group consisting of MenA, MenC, MenY and MenW which is/are conjugated through a linker to a carrier protein(s), and one or more different saccharides is/are selected from a second group consisting of MenA, MenC, MenY and MenW which is/are directly conjugated to a carrier protein(s).

The present invention relates to immunogenic compositions comprisingbacterial capsular saccharides conjugated to a carrier protein, inparticular those saccharides of N. meningitidis. It additionally relatesto vaccines and vaccine kits comprising such saccharide conjugates,processes for making the immunogenic compositions and vaccines and theuse of the vaccines and immunogenic compositions of the invention intherapy. It also relates to methods of immunising against infectionusing the saccharide conjugates and the use of the saccharide conjugatesin the manufacture of a medicament.

Neisseria meningitidis is a Gram-negative human pathogen which causesbacterial meningitis. Based on the organism's capsular polysaccharide,twelve serogroups of N. meningitidis have been identified (A, B, C, H,I, K, L, 29E, W135, X, Y and Z). Serogroup A (MenA) is the most commoncause of epidemic disease in sub-Saharan Africa. Serogroups B and C areresponsible for the majority of cases in developing countries, with theremaining cases being caused by W135 and Y).

Immunogenic compositions comprising N. meningitidis saccharidesconjugated to carrier proteins are known in the art; the carrier proteinhaving the known effect of turning the T-independent polysaccharideantigen into a T-dependent antigen capable of triggering an immunememory response. For instance WO 02/58737 discloses a vaccine comprisingpurified capsular polysaccharides from N. meningitidis serogroups A, C,W135 and Y conjugated to a carrier protein. However, this applicationteaches that all polysaccharides should essentially be conjugated in thesame way (through the same linker to the same protein carrier).

There remains a need to develop improved conjugate vaccines againstneisserial meningitis. The present invention concerns the provision of ameningococcal polysaccharide conjugate vaccine where conjugation of eachpolysaccharide is tailored (rather than being uniform) to achieve anefficacious combination vaccine.

In particular it is advantageous to use linker molecules to conjugatecertain meningococcal saccharides to their protein carriers incombination with others that are directly conjugated. In this waypolysaccharides that are less good immunogens may be presented to theimmune system via a linker, and those that are very good immunogens maybe directly conjugated so that they do not dominate the immune responseto the combination.

Accordingly, in one aspect of the present invention there is provided animmunogenic composition comprising at least 2 different N. meningitidiscapsular saccharides, wherein one or more is/are selected from a firstgroup consisting of MenA, MenC, MenY and MenW which is/are conjugatedthrough a linker to a carrier protein(s), and one or more differentsaccharides is/are selected from a second group consisting of MenA,MenC, MenY and MenW which is/are directly conjugated to a carrierprotein(s).

In a MenAC vaccine, for example, MenA may be conjugated through a linkerand MenC directly. In a MenCY vaccine, MenC may be conjugated through alinker and MenY directly. In a MenACWY vaccine Men A may be conjugatedthrough a linker and MenCWY directly, or MenAC may be conjugated througha linker and MenWY directly.

A further consideration in a combination vaccine comprising varioussaccharides conjugated to the same carrier is the issue of carrierimmune suppression: too much carrier may be used and the immune responsemay be dampened. With a uniform approach to conjugation the carrier willpresent a similar blend of B- and T-cell epitopes to the immune system.However if conjugation takes place at different chemical groups withinthe carrier protein for one saccharide versus another, the proteincarriers are likely to be different to some extent in how they presentthemselves to the immune system.

Accordingly, in a separate embodiment of the invention there is providedan immunogenic composition comprising at least 2 different saccharidesconjugated separately to the same type of carrier protein (for instancetetanus toxoid), wherein one or more saccharide(s) is/are conjugated tothe carrier protein via a first type of chemical group on the proteincarrier, and one or more saccharide(s) is/are conjugated to the carrierprotein via a second (different) type of chemical group on the proteincarrier.

The first and second types of chemical group may be present in theprotein carrier on a mutually exclusive first and second set of aminoacids of the protein carrier (for instance certain asparticacid/glutamic acid residues in one set and certain lysine residues inthe second). One saccharide may be conjugated to a carboxyl group on thecarrier, and another on an amino group for instance. Such conjugationmay involve conjugation on separate B- and/or T-cell epitopes for eachdifferent conjugate.

For instance in a MenAC vaccine, MenA may be linked to a first type ofchemical group (such as carboxyl) on the carrier protein and MenC linkedto a second (such as amino). In a MenCY vaccine MenC may be linked to afirst type of chemical group (such as carboxyl) on the carrier proteinand MenY linked to a second (such as amino). In a MenACWY vaccine, MenACmay be linked to a first type of chemical group (such as carboxyl) onthe carrier protein and MenWY linked to a second (such as amino), orMenA may be linked to a first type of chemical group (such as carboxyl)on the carrier protein and MenCWY linked to a second (such as amino).

According to a further aspect of the invention there is provided amethod of immunising a human host against disease caused by Neisseriameningitidis comprising administering to the host an immunoprotectivedose of the immunogenic composition or vaccine of the invention.

According to a further aspect or the invention there is provided animmunogenic composition of the invention for use in the treatment orprevention of disease caused by Neisseria meningitidis.

According to a further aspect or the invention there is provided a useof the immunogenic composition or vaccine of the invention in themanufacture of a medicament for the treatment or prevention of diseasescaused by Neisseria meningitidis.

DESCRIPTION OF FIGURES

FIG. 1—A—Bar chart showing GMC responses in an anti-MenY ELISA. ENYTT012is a MenY-TT conjugate prepared from native MenY polysaccharide.ENYTT014 is a MenY-TT conjugate prepared from microfluidised MenYpolysaccharide which had undergone 40 cycles of microfluidisation.ENYTT015bis is a MenY-TT conjugate prepared from microfluidised MenYpolysaccharide which had undergone 20 cycles of microfluidisation.

B—Bar chart showing GMT responses in an anti-MenY SBA assay. ENYTT012 isa MenY-TT conjugate prepared from native MenY polysaccharide. ENYTT014is a MenY-TT conjugate prepared from microfluidised MenY polysaccharidewhich had undergone 40 cycles of microfluidisation. ENYTT015bis is aMenY-TT conjugate prepared from microfluidised MenY polysaccharide whichhad undergone 20 cycles of microfluidisation.

DETAILED DESCRIPTION

In one aspect of the present invention there is provided an immunogeniccomposition comprising at least 2 different N. meningitidis capsularsaccharides, wherein one or more is/are selected from a first groupconsisting of MenA, MenC, MenY and MenW which is/are conjugated througha linker to a carrier protein(s), and one or more different saccharidesis/are selected from a second group consisting of MenA, MenC, MenY andMenW which is/are directly conjugated to a carrier protein(s).

More specifically, the first group may consist of MenA and MenC, and thesecond group consist of MenC, MenY and MenW. Particular embodiments ofthe invention are immunogenic compositions comprising: MenA capsularsaccharide conjugated through a linker to a carrier protein and MenCcapsular saccharide directly conjugated to a carrier protein; MenCcapsular saccharide conjugated through a linker to a carrier protein andMenY capsular saccharide directly conjugated to a carrier protein; MenAand MenC capsular saccharides conjugated through a linker to a carrierprotein(s) and MenY and Men W capsular saccharides directly conjugatedto a carrier protein(s); MenA capsular saccharide conjugated through alinker to a carrier protein and MenC, MenY and Men W capsularsaccharides directly conjugated to a carrier protein(s). In any of theseembodiments a Hib conjugate may also be included, which is linked to acarrier protein (see list of carriers above and below, for example TT)directly or through a linker.

The term “saccharide” throughout this specification may indicatepolysaccharide or oligosaccharide and includes both. Polysaccharides areisolated from bacteria or isolated from bacteria and sized to somedegree by known methods (see for example EP497524 and EP497525) andoptionally by microfluidisation. Polysaccharides can be sized in orderto reduce viscosity in polysaccharide samples and/or to improvefilterability for conjugated products. Oligosaccharides have a lownumber of repeat units (typically 5-30 repeat units) and are typicallyhydrolysed polysaccharides.

Each N. meningitidis (and/or Hib) capsular saccharide may be conjugatedto a carrier protein independently selected from the group consisting ofTT, DT, CRM197, fragment C of TT and protein D. A more complete list ofprotein carriers that may be used in the conjugates of the invention ispresented below. Although one or more N. meningitidis (and/or Hib)capsular saccharide may be conjugated to different carrier proteins fromthe others, in one embodiment they are all conjugated to the samecarrier protein. For instance they may all be conjugated to the samecarrier protein selected from the group consisting of TT, DT, CRM197,fragment C of TT and protein D. In this context CRM197 and DT may beconsidered to be the same carrier protein as they differ by only oneamino acid. In an embodiment all the N. meningitidis (and/or Hib)capsular saccharides present are conjugated to TT.

If the protein carrier is the same for 2 or more saccharides in thecomposition, the saccharide could be conjugated to the same molecule ofthe protein carrier (carrier molecules having 2 more differentsaccharides conjugated to it) [see for instance WO 04/083251; forexample, a single carrier protein might be conjugated to MenA and MenC;MenA and MenW; MenA and MenY; MenC and MenW; MenC and MenY; Men W andMenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA, MenW and MenY;MenC, MenW and MenY; MenA, MenC, MenW and MenY; Hib and MenA; Hib andMenC; Hib and MenW; or Hib and MenY]. Alternatively the saccharides mayeach be separately conjugated to different molecules of the proteincarrier (each molecule of protein carrier only having one type ofsaccharide conjugated to it).

Immunogenic compositions of the first aspect of the invention may alsohave any or all the additional characteristics of the second aspect ofthe invention and vice versa.

In a second aspect of the invention there is presented an immunogeniccomposition comprising at least 2 different saccharide conjugatesconjugated separately to the same type of carrier protein, wherein oneor more saccharide(s) is/are conjugated to the carrier protein via afirst type of chemical group on the protein carrier, and one or moresaccharide(s) is/are conjugated to the carrier protein via a second(different) type of chemical group on the protein carrier.

In one embodiment the 2 conjugates involve the same saccharide linked tothe same carrier, but by different conjugation chemistries. In analternative embodiment 2 different saccharides are conjugated todifferent groups on the protein carrier.

By “conjugated separately to the same type of carrier protein” it ismeant that the saccharides are conjugated to the same carrierindividually (for example, MenA is conjugated to tetanus toxoid throughan amine group on the tetanus toxoid and MenC is conjugated to tetanustoxoid through a carboxylic acid group on a different molecule oftetanus toxoid.)

The capsular saccharide(s) may be conjugated to the same carrier proteinindependently selected from the group consisting of TT, DT, CRM197,fragment C of TT and protein D. A more complete list of protein carriersthat may be used in the conjugates of the invention is presented below.In this context CRM197 and DT may be considered to be the same carrierprotein as they differ by only one amino acid. In an embodiment all thecapsular saccharides present are conjugated to TT.

In one embodiment the first and second type of chemical group on theprotein carrier are present on separate B- and/or T-cell epitopes on thecarrier protein. That is, they are present on a different set of B-and/or T-cell epitopes from each other. To predict B-cell epitopes for acarrier known methods may be used such as either or both of thefollowing two methods: 2D-structure prediction and/or antigenic indexprediction. 2D-structure prediction can be made using the PSIPREDprogram (from David Jones, Brunel Bioinformatics Group, Dept. BiologicalSciences, Brunel University, Uxbridge UB8 3PH, UK). The antigenic indexcan be calculated on the basis of the method described by Jameson andWolf (CABIOS 4:181-186 [1988]). The parameters used in this program arethe antigenic index and the minimal length for an antigenic peptide. Anantigenic index of 0.9 for a minimum of 5 consecutive amino acids can beused as the thresholds in the program. T-helper cell epitopes arepeptides bound to HLA class II molecules and recognized by T-helpercells. The prediction of useful T-helper cell epitopes can be based onknown techniques, such as the TEPITOPE method describe by Sturniolo atal. (Nature Biotech. 17: 555-561 [1999]).

The saccharides may be selected from a group consisting of: N.meningitidis serogroup A capsular saccharide (MenA), N. meningitidisserogroup C capsular saccharide (MenC), N. meningitidis serogroup Ycapsular saccharide (MenY), N. meningitidis serogroup W capsularsaccharide (MenW), H. influenzae type b capsular saccharide (Hib), GroupB Streptococcus group I capsular saccharide, Group B Streptococcus groupII capsular saccharide, Group B Streptococcus group III capsularsaccharide, Group B Streptococcus group IV capsular saccharide, Group BStreptococcus group V capsular saccharide, Staphylococcus aureus type 5capsular saccharide, Staphylococcus aureus type 8 capsular saccharide,Vi saccharide from Salmonella typhi, N. meningitidis LPS (such as L3and/or L2), M. catarrhalis LPS, H. influenzae LPS, and from any of thecapsular pneumococcal saccharides such as from serotype: 1, 2, 3, 4, 5,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F or 33F. In one embodiment the immunogenic composition of theinvention consists of or comprises two or more different saccharidesfrom the same genus of bacteria (e.g. Neisseria, Streptococcus,Staphylococcus, or Haemophilus).

The first and second chemical groups present on the protein carrier aredifferent from each other and are ideally natural chemical groups thatmay be readily used for conjugation purposes. They may be selectedindependently from the group consisting of: carboxyl groups, aminogroups, sulphydryl groups, Hydroxyl groups, Imidazolyl groups, Guanidylgroups, and Indolyl groups. In one embodiment the first chemical groupis carboxyl and the second is amino, or vice versa. These groups areexplained in greater detail below.

In a specific embodiment the immunogenic composition comprises at least2 different N. meningitidis capsular saccharides, wherein one or moreis/are selected from a first group consisting of MenA and MenC whichis/are conjugated to the carrier protein via the first type of chemicalgroup on the protein carrier (for instance carboxyl), and one or moredifferent saccharides is/are selected from a second group consisting ofMenC, MenY and MenW which is/are conjugated to the carrier protein viathe second type of chemical group on the protein carrier (for instanceamino).

In a further embodiment the immunogenic composition of the inventioncomprises MenA conjugated via the first type of chemical group (forinstance carboxyl), and MenC conjugated via the second type of chemicalgroup (for instance amino).

In another embodiment the immunogenic composition comprises MenCconjugated via the first type of chemical group (for instance carboxyl),and MenY conjugated via the second type of chemical group (for instanceamino).

In another embodiment the immunogenic composition comprises MenAconjugated via the first type of chemical group (for instance carboxyl),and MenC, MenY and MenW conjugated via the second type of chemical group(for instance amino).

In another embodiment the immunogenic composition comprises MenA andMenC conjugated via the first type of chemical group (for instancecarboxyl), and MenY and MenW conjugated via the second type of chemicalgroup (for instance amino).

In any of the above embodiments Hib may also be present also conjugatedto the same type of protein carrier. Hib may be conjugated to thecarrier by the first or second type of chemical group. In one embodimentit is conjugated via a carboxyl group.

General Considerations in the Aspects of the Invention

The saccharides of the invention (in particular the N. meningitidissaccharides and/or the Hib capsular saccharide) included inpharmaceutical (immunogenic) compositions of the invention areconjugated to a carrier protein such as tetanus toxoid (TT), tetanustoxoid fragment C, non-toxic mutants of tetanus toxin [note all suchvariants of TT are considered to be the same type of carrier protein forthe purposes of this invention], diphtheria toxoid (DT), CRM197, othernon-toxic mutants of diphtheria toxin [such as CRM176, CRM 197, CRM228,CRM 45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM45, CRM102, CRM 103 and CRM107 and other mutations described by Nichollsand Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel DekkerInc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala158 to Gly and other mutations disclosed in U.S. Pat. No. 4,709,017 orU.S. Pat. No. 4,950,740; mutation of at least one or more residues Lys516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed inU.S. Pat. No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragmentdisclosed in U.S. Pat. No. 5,843,711] (note all such variants of DT areconsidered to be the same type of carrier protein for the purposes ofthis invention), pneumococcal pneumolysin (Kuo et al (1995) Infect Immun63; 2706-13), OMPC (meningococcal outer membrane protein—usuallyextracted from N. meningitidis serogroup B—EP0372501), syntheticpeptides (EP0378881, EPO427347), heat shock proteins (WO 93/17712, WO94/03208), pertussis proteins (WO 98/58668, EPO471177), cytokines,lymphokines, growth factors or hormones (WO 91/01146), artificialproteins comprising multiple human CD4+ T cell epitopes from variouspathogen derived antigens (Falugi et al (2001) Eur J Immunol 31;3816-3824) such as N19 protein (Baraldoi et al (2004) Infect Immun 72;4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptakeproteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761) orProtein D (EP594610 and WO 00/56360).

In an embodiment, the immunogenic composition of the invention uses thesame type of carrier protein (independently) in at least two, three,four or each of the saccharides (e.g. N. meningitidis capsularsaccharides and/or Hib) contained therein. In an embodiment where Hiband N. meningitidis capsular saccharides are present, Hib may beconjugated to the same type of carrier protein as the at least two,three, four or each of the N. meningitidis saccharides. For example, 2,3 or 4 of the N. meningitidis saccharides (MenA,C,Y,W) are independentlyconjugated to tetanus toxoid to make 2, 3 or 4 conjugates, andoptionally Hib is also conjugated to TT.

In an embodiment, the immunogenic composition of the invention comprisesa N. meningitidis saccharide conjugated to a carrier protein selectedfrom the group consisting of TT, DT, CRM197, fragment C of TT andprotein D. In an embodiment, the immunogenic composition of theinvention comprises a Hib saccharide conjugated to a carrier proteinselected from the group consisting of TT, DT, CRM197, fragment C of TTand protein D.

The immunogenic composition of the invention optionally comprises atleast one meningococcal saccharide (for example MenA; MenC; MenW; MenY;MenA and MenC; MenA and MenW; MenA and MenY; MenC and Men W; Men C andMenY; Men W and MenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA,MenW and MenY; MenC, MenW and MenY or MenA, MenC, MenW and MenY)conjugate having a ratio of Men saccharide to carrier protein of between1:5 and 5:1, between 1:2 and 5:1, between 1:0.5 and 1:2.5 or between1:1.25 and 1:2.5 (w/w).

The immunogenic composition of the invention optionally comprises a Hibsaccharide conjugate having a ratio of Hib to carrier protein of between1:5 and 5:1; 1:2 and 2:1; 1:1 and 1:4; 1:2 and 1:3.5; or around orexactly 1:2.5 or 1:3 (w/w).

The ratio of saccharide to carrier protein (w/w) in a conjugate may bedetermined using the sterilized conjugate. The amount of protein isdetermined using a Lowry assay (for example Lowry et al (1951) J. Biol.Chem. 193, 265-275 or Peterson et al Analytical Biochemistry 100,201-220 (1979)) and the amount of saccharide is determined using ICP-OES(inductively coupled plasma-optical emission spectroscopy) for MenA,DMAP assay for MenC and Resorcinol assay for MenW and MenY (Monsigny etal (1988) Anal. Biochem. 175, 525-530).

In an embodiment, the immunogenic composition of the invention comprisesN. meningitidis saccharide conjugate(s) and/or the Hib saccharideconjugate wherein the N. meningitidis saccharide(s) and/or the Hibsaccharide is conjugated to the carrier protein via a linker, forinstance a bifunctional linker. The linker is optionallyheterobifunctional or homobifunctional, having for example a reactiveamino group and a reactive carboxylic acid group, 2 reactive aminogroups or two reactive carboxylic acid groups. The linker has forexample between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possiblelinker is ADH. Other linkers include B-propionamido (WO 00/10599),nitrophenyl-ethylamine (Geyer et al (1979) Med. Microbiol. Immunol. 165;171-288), haloalkyl halides (U.S. Pat. No. 4,057,685), glycosidiclinkages (U.S. Pat. No. 4,673,574, U.S. Pat. No. 4,808,700), hexanediamine and 6-aminocaproic acid (U.S. Pat. No. 4,459,286).

The saccharide conjugates present in the immunogenic compositions of theinvention may be prepared by any known coupling technique. Theconjugation method may rely on activation of the saccharide with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated saccharide may thus be coupled directly orvia a spacer (linker) group to an amino group on the carrier protein.For example, the spacer could be cystamine or cysteamine to give athiolated polysaccharide which could be coupled to the carrier via athioether linkage obtained after reaction with a maleimide-activatedcarrier protein (for example using GMBS) or a holoacetylated carrierprotein (for example using iodoacetimide or N-succinimidylbromoacetatebromoacetate). Optionally, the cyanate ester (optionallymade by CDAP chemistry) is coupled with hexane diamine or ADH and theamino-derivatised saccharide is conjugated to the carrier protein usingusing carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group onthe protein carrier. Such conjugates are described in PCT publishedapplication WO 93/15760 Uniformed Services University and WO 95/08348and WO 96/29094.

Other suitable techniques use carbiinides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in WO 98/42721. Conjugation may involve a carbonyllinker which may be formed by reaction of a free hydroxyl group of thesaccharide with CDI (Bethell et al J. Biol. Chem. 1979, 254; 2572-4,Hearn et al J. Chromatogr. 1981. 218; 509-18) followed by reaction ofwith a protein to form a carbamate linkage. This may involve reductionof the anomeric terminus to a primary hydroxyl group, optionalprotection/deprotection of the primary hydroxyl group' reaction of theprimary hydroxyl group with CDI to form a CDI carbamate intermediate andcoupling the CDI carbamate intermediate with an amino group on aprotein.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508.

A further method involves the coupling of a cyanogen bromide (or CDAP)activated saccharide derivatised with adipic acid hydrazide (ADH) to theprotein carrier by Carbodiimide condensation (Chu C. et al Infect.Immunity, 1983 245 256), for example using EDAC.

In an embodiment, a hydroxyl group (optionally an activated hydroxylgroup for example a hydroxyl group activated by a cyanate ester) on asaccharide is linked to an amino or carboxylic group on a protein eitherdirectly or indirectly (through a linker). Where a linker is present, ahydroxyl group on a saccharide is optionally linked to an amino group ona linker, for example by using CDAP conjugation. A further amino groupin the linker for example ADH) may be conjugated to a carboxylic acidgroup on a protein, for example by using carbodiimide chemistry, forexample by using EDAC. In an embodiment, the Hib or N. meningitidiscapsular saccharide(s) (or saccharide in general) is conjugated to thelinker first before the linker is conjugated to the carrier protein.Alternatively the linker may be conjugated to the carrier beforeconjugation to the saccharide.

In general the following types of chemical groups on a protein carriercan be used for coupling/conjugation:

A) Carboxyl (for instance via aspartic acid or glutamic acid). In oneembodiment this group is linked to amino groups on saccharides directlyor to an amino group on a linker with carbodiimide chemistry e.g. withEDAC.

B) Amino group (for instance via lysine). In one embodiment this groupis linked to carboxyl groups on saccharides directly or to a carboxylgroup on a linker with carbodiimide chemistry e.g. with EDAC. In anotherembodiment this group is linked to hydroxyl groups activated with CDAPor CNBr on saccharides directly or to such groups on a linker; tosaccharides or linkers having an aldehyde group; to saccharides orlinkers having a succinimide ester group.

C) Sulphydryl (for instance via cysteine). In one embodiment this groupis linked to a bromo or chloro acetylated saccharide or linker withmaleimide chemistry. In one embodiment this group is activated/modifiedwith bis diazobenzidine.

D) Hydroxyl group (for instance via tyrosine). In one embodiment thisgroup is activated/modified with bis diazobenzidine.

E) Imidazolyl group (for instance via histidine). In one embodiment thisgroup is activated/modified with bis diazobenzidine.

F) Guanidyl group (for instance via arginine).

G) Indolyl group (for instance via tryptophan).

On a saccharide, in general the following groups can be used for acoupling: OH, COOH or NH2. Aldehyde groups can be generated afterdifferent treatments known in the art such as: periodate, acidhydrolysis, hydrogen peroxide, etc.

Direct Coupling Approaches:

Saccharide-OH+CNBr or CDAP---->cyanate ester+NH2-Prot---->conjugate

Saccharide-aldehyde+NH2-Prot---->Schiff base+NaCNBH3---->conjugate

Saccharide-COOH+NH2-Prot+EDAC---->conjugate

Saccharide-NH2+COOH-Prot+EDAC---->conjugate

Indirect Coupling Via Spacer (Linker) Approaches:

Saccharide-OH+CNBr or CDAP--->cyanateester+NH2----NH2---->saccharide----NH2+COOH-Prot+EDAC---->conjugate

Saccharide-OH+CNBr or CDAP---->cyanate ester+NH2SH------>saccharide----SH+SH-Prot (native Protein with an exposedcysteine or obtained after modification of amino groups of the proteinby SPDP for instance)---->saccharide-S-S-Prot

Saccharide-OH+CNBr or CDAP--->cyanate ester+NH2----SHsaccharide----SH+maleimide-Prot (modification of aminogroups)---->conjugate

Saccharide-COOH+EDAC+NH2 NH2--->saccharideNH2+EDAC+COOH-Prot---->conjugate

Saccharide-COOH+EDAC+NH2----SH---->saccharide----SH+SH-Prot (nativeProtein with an exposed cysteine or obtained after modification of aminogroups of the protein by SPDP for instance)----->saccharide-S-S-Prot

Saccharide-COOH+EDAC+NH2----SH----->saccharide----SH+maleimide-Prot(modification of amino groups)---->conjugate

Saccharide-Aldehyde+NH2NH2---->saccharide---NH2+EDAC+COOH-Prot---->conjugate

Note: instead of EDAC above, any suitable carbodiimide may be used.

In summary, the types of protein carrier chemical group that may begenerally used for coupling with a saccharide are amino groups (forinstance on lysine residues), COOH groups (for instance on aspartic andglutamic acid residues) and SH groups (if accessible) (for instance oncysteine residues).

In an embodiment, the Hib saccharide, where present, is conjugated tothe carrier protein using CNBr, or CDAP, or a combination of CDAP andcarbodiimide chemistry (such as EDAC), or a combination of CNBr andcarbodiimide chemistry (such as EDAC). Optionally Hib is conjugatedusing CNBr and carbodiimide chemistry, optionally EDAC. For example,CNBr is used to join the saccharide and linker and then carbodiimidechemistry is used to join linker to the protein carrier.

In an embodiment, at least one of the N. meningitidis capsularsaccharides (or saccharide in general) is directly conjugated to acarrier protein; optionally Men W and/or MenY and/or MenC saccharide(s)is directly conjugated to a carrier protein. For example MenW; MenY;MenC; MenW and MenY; MenW and MenC; MenY and MenC; or MenW, MenY andMenC are directly linked to the carrier protein. Optionally, at leastone of the N. meningitidis capsular saccharides is directly conjugatedby CDAP. For example MenW; MenY; MenC; MenW and MenY; MenW and MenC;MenY and MenC; or MenW, MenY and MenC are directly linked to the carrierprotein by CDAP (see WO 95/08348 and WO 96/29094). In an embodiment, allN. meningitidis capsular saccharides are conjugated to tetanus toxoid.

In an embodiment, the ratio of Men W and/or Y saccharide to carrierprotein is between 1:0.5 and 1:2 (w/w) and/or the ratio of MenCsaccharide to carrier protein is between 1:0.5 and 1:4 or 1:0.5 and1:1.5 (w/w), especially where these saccharides are directly linked tothe protein, optionally using CDAP.

In an embodiment, at least one of the N. meningitidis capsularsaccharide(s) (or saccharide in general) is conjugated to the carrierprotein via a linker, for instance a bifunctional linker. The linker isoptionally heterobifunctional or homobifunctional, having for example areactive amine group and a reactive carboxylic acid group, 2 reactiveamine groups or 2 reactive carboxylic acid groups. The linker has forexample between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possiblelinker is ADH.

In an embodiment, MenA; MenC; or MenA and MenC is conjugated to acarrier protein (for example tetanus toxoid) via a linker.

In an embodiment, at least one N. meningitidis saccharide is conjugatedto a carrier protein via a linker using CDAP and EDAC. For example,MenA; MenC; or MenA and MenC are conjugated to a protein via a linker(for example those with two hydrazino groups at its ends such as ADH)using CDAP and EDAC as described above. For example, CDAP is used toconjugate the saccharide to a linker and EDAC is used to conjugate thelinker to a protein. Optionally the conjugation via a linker results ina ratio of saccharide to carrier protein of of between 1:0.5 and 1:6;1:1 and 1:5 or 1:2 and 1:4, for MenA; MenC; or MenA and MenC.

In an embodiment, the MenA capsular saccharide, where present is atleast partially O-acetylated such that at least 50%, 60%, 70%, 80%, 90%,95% or 98% of the repeat units are O-acetylated at at least oneposition. O-acetylation is for example present at least at the O-3position of at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeatunits.

In an embodiment, the MenC capsular saccharide, where present is is atleast partially O-acetylated such that at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or 98% of (α2→9)-linked NeuNAc repeat units areO-acetylated at at least one or two positions. O-acetylation is forexample present at the O-7 and/or O-8 position of at least 30%. 40%,50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.

In an embodiment, the MenW capsular saccharide, where present is is atleast partially O-acetylated such that at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or 98% of the repeat units are O-acetylated at at leastone or two positions. 0-acetylation is for example present at the O-7and/or O-9 position of at least 30%. 40%, 50%, 60%, 70%, 80%, 90%, 95%or 98% of the repeat units.

In an embodiment, the MenY capsular saccharide, where present is atleast partially O-acetylated such that at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% or 98% of the repeat units are O-acetylated at atleast one or two positions. O-acetylation is present at the 7 and/or 9position of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98%of the repeat units.

The percentage of O-acetylation refers to the percentage of the repeatunits containing O-acetylation. This may be measured in the saccharideprior to conjugate and/or after conjugation.

In one embodiment of the invention the immunogenic composition,saccharide present, or each N. meningitidis capsular saccharide present,is conjugated to TT. In a further embodiment each N. meningitidiscapsular saccharide is separately conjugated to a separate carrierprotein. In a further embodiment each N. meningitidis capsularsaccharide conjugate has a saccharide:carrier ratio of 1:5-5:1 or1:1-1:4 (w/w). In a further embodiment at least one, two or three N.meningitidis capsular saccharide conjugate(s) is directly conjugated toa carrier protein. In a further embodiment Men W and/or MenY, MenWand/or MenC, MenY and/or MenC, or MenW and MenC and MenY are directlyconjugated to a carrier protein. In a further embodiment at least one,two or three N. meningitidis saccharide conjugate(s) is directlyconjugated by CDAP chemistry. In a further embodiment the ratio of Men Wand/or Y saccharide to carrier protein is between 1:0.5 and 1:2 (w/w).In a further embodiment the ratio of MenC saccharide to carrier proteinis between 1:0.5 and 1:2 (w/w). In a further embodiment at least one,two or three N. meningitidis capsular saccharide(s) are conjugated tothe carrier protein via a linker (which may be bifunctional such ashaving two reactive amino groups (such as ADH) or two reactive carboxylgroups, or a reactive amino group at one end and a reactive carboxylgroup at the other). The linker can have between 4 and 12 carbon atoms.In a further embodiment the or each N. meningitidis capsularsaccharide(s) conjugated via a linker are conjugated to the linker withCDAP chemistry. In a further embodiment the carrier protein isconjugated to the linker using carbodiimide chemistry, for example usingEDAC. In a further embodiment the or each N. meningitidis capsularsaccharide is conjugated to the linker before the carrier protein isconjugated to the linker. In a further embodiment MenA is conjugated toa carrier protein via a linker (the ratio of MenA saccharide to carrierprotein may be between 1:2 and 1:5 (w/w)). In a further embodiment MenCis conjugated to a carrier protein via a linker (the ratio of MenCsaccharide to carrier protein may be between 1:2 and 1:5 (w/w)).

The inventors have also noted that the focus of the art has been to useoligosaccharides for ease of conjugate production. The inventors havefound that by using native or slightly sized polysaccharide conjugates,one or more of the following advantages may be realised: 1) a conjugatehaving high immunogenicity which is filterable through a 0.2 micronfilter; 2) immune memory may be enhanced (as in example three); 3) thealteration of the ratio of polysaccharide to protein in the conjugatesuch that the ratio of polysaccharide to protein (w/w) in the conjugatemay be increased (this can result in a reduction of the carriersuppression effect); 4) immunogenic conjugates prone to hydrolysis (suchas MenA conjugates) may be stabilised by the use of largerpolysaccharides for conjugation. The use of larger polysaccharides canresult in more cross-linking with the conjugate carrier and may lessenthe liberation of free saccharide from the conjugate. The conjugatevaccines described in the prior art tend to depolymerise thepolysaccharides prior to conjugation in order to improve conjugation.The present inventors have found that meningococcal (or saccharide)conjugate vaccines retaining a larger size of saccharide can provide agood immune response against meningococcal disease.

The immunogenic composition of the invention may thus comprise one ormore saccharide conjugates wherein the average size of each saccharidebefore conjugation is above 50 kDa, 75 kDa, 100 kDa, 110 kDa, 120 kDa or130 kDa. In one embodiment the conjugate post conjugation should bereadily filterable through a 0.2 micron filter such that a yield of morethan 50, 60, 70, 80, 90 or 95% is obtained post filtration compared withthe pre filtration sample.

In particular, the immunogenic composition of the invention comprises N.meningitidis capsular saccharides from at least one, two, three or fourof serogroups A, C, W and Y conjugated to a carrier protein, wherein theaverage size (weight-average molecular weight; Mw) of at least one, two,three or four or each N. meningitidis saccharide is above 50 kDa, 60kDa, 75 kDa, 100 kDa, 110 kDa, 120 kDa or 130 kDa.

The immunogenic composition may comprise N. meningitidis capsularsaccharides from at least one, two, three or four of serogroups A, C, Wand Y conjugated to a carrier protein, wherein at least one, two, threeor four or each N. meningitidis saccharide is either a native saccharideor is sized by a factor up to ×2, ×3, ×4, ×5, ×6, ×7, ×8, ×9 or ×10relative to the weight average molecular weight of the nativepolysaccharide.

For the purposes of the invention, “native polysaccharide” refers to asaccharide that has not been subjected to a process, the purpose ofwhich is to reduce the size of the saccharide. A polysaccharide canbecome slightly reduced in size during normal purification procedures.Such a saccharide is still native. Only if the polysaccharide has beensubjected to sizing techniques would the polysaccharide not beconsidered native.

For the purposes of the invention, “sized by a factor up to ×2” meansthat the saccharide is subject to a process intended to reduce the sizeof the saccharide but to retain a size more than half the size of thenative polysaccharide. ×3, ×4 etc. are to be interpreted in the same wayi.e. the saccharide is subject to a process intended to reduce the sizeof the polysaccharide but to retain a size more than a third, a quarteretc. the size of the native polysaccharide.

In an aspect of the invention, the immunogenic composition comprises N.meningitidis capsular saccharides from at least one, two, three or fourof serogroups A, C, W and Y conjugated to a carrier protein, wherein atleast one, two, three or four or each N. meningitidis saccharide isnative polysaccharide.

In an aspect of the invention, the immunogenic composition comprises N.meningitidis capsular saccharides from at least one, two, three or fourof serogroups A, C, W and Y conjugated to a carrier protein, wherein atleast one, two, three or four or each N. meningitidis saccharide issized by a factor up to ×1.5, ×2, ×3, ×4, ×5, ×6, ×7, ×8, ×9 or ×10.

The immunogenic compositions of the invention optionally compriseconjugates of: N. meningitidis serogroup C capsular saccharide (MenC),serogroup A capsular saccharide (MenA), serogroup W135 capsularsaccharide (MenW), serogroup Y capsular saccharide (MenY), serogroup Cand Y capsular saccharides (MenCY), serogroup C and A capsularsaccharides (MenAC), serogroup C and W capsular saccharides (MenCW),serogroup A and Y capsular saccharide (MenAY), serogroup A and Wcapsular saccharides (MenAW), serogroup W and Y capsular saccharides(Men WY), serogroup A, C and W capsular saccharide (MenACW), serogroupA, C and Y capsular saccharides (MenACY); serogroup A, W135 and Ycapsular saccharides (MenAWY), serogroup C, W135 and Y capsularsaccharides (MenCWY); or serogroup A, C, W135 and Y capsular saccharides(MenACWY). This is the definition of “one, two, three or four”, or “atleast one of” of serogroups A, C, W and Y, or of each N. meningitidissaccharide where mentioned herein.

In an embodiment, the average size of at least one, two, three, four oreach N. meningitidis saccharide is between 50 KDa and 1500 kDa, 50 kDaand 500 kDa, 50 kDa and 300 KDa, 101 kDa and 1500 kDa, 101 kDa and 500kDa, 101 kDa and 300 kDa as determined by MALLS.

In an embodiment, the MenA saccharide, where present, has a molecularweight of 50-500 kDa, 50-100 kDa, 100-500 kDa, 55-90 KDa, 60-70 kDa or70-80 kDa or 60-80 kDa.

In an embodiment, the MenC saccharide, where present, has a molecularweight of 100-200 kDa, 50-100 kDa, 100-150 kDa, 101-130 kDa, 150-210 kDaor 180-210 kDa.

In an embodiment the MenY saccharide, where present, has a molecularweight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa, 100-150 kDa or110-140 kDa, 50-100 kDa, 100-140 kDa, 140-170 kDa or 150-160 kDa.

In an embodiment the MenW saccharide, where present, has a molecularweight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa, 100-150 kDa,110-140 kDa, 50-100 kDa or 120-140 kDa.

The molecular weight or average molecular weight of a saccharide hereinrefers to the weight-average molecular weight (Mw) of the saccharidemeasured prior to conjugation and is measured by MALLS.

The MALLS technique is well known in the art and is typically carriedout as described in example 2. For MALLS analysis of meningococcalsaccharides, two columns (TSKG6000 and 5000PWxl) may be used incombination and the saccharides are eluted in water. Saccharides aredetected using a light scattering detector (for instance Wyatt Dawn DSPequipped with a 10 mW argon laser at 488 nm) and an inferometricrefractometer (for instance Wyatt Otilab DSP equipped with a P100 celland a red filter at 498 nm).

In an embodiment the N. meningitidis saccharides are nativepolysaccharides or native polysaccharides which have reduced in sizeduring a normal extraction process.

In an embodiment, the N. meningitidis saccharides are sized bymechanical cleavage, for instance by microfluidisation or sonication.Microfluidisation and sonication have the advantage of decreasing thesize of the larger native polysaccharides sufficiently to provide afilterable conjugate (fro example through a 0.2 micron filter). Sizingis by a factor of no more than ×20, ×10, ×8, ×6, ×5, ×4, ×3, ×2 or ×1.5.

In an embodiment, the immunogenic composition comprises N. meningitidisconjugates that are made from a mixture of native polysaccharides andsaccharides that are sized by a factor of no more than ×20. For example,saccharides from MenC and/or MenA are native. For example, saccharidesfrom MenY and/or MenW are sized by a factor of no more than ×20, ×10,×8, ×6, ×5, ×4, ×3 or ×2. For example, an immunogenic compositioncontains a conjugate made from MenY and/or MenW and/or MenC and/or MenAwhich is sized by a factor of no more then ×10 and/or is microfluidised.For example, an immunogenic composition contains a conjugate made fromnative MenA and/or MenC and/or MenW and/or MenY. For example, animmunogenic composition comprises a conjugate made from native MenC. Forexample, an immunogenic composition comprises a conjugate made fromnative MenC and MenA which is sized by a factor of no more then ×10and/or is microfluidised. For example, an immunogenic compositioncomprises a conjugate made from native MenC and MenY which is sized by afactor of no more then ×10 and/or is microfluidised.

In an embodiment, the polydispersity of the saccharide is 1-1.5, 1-1.3,1-1.2, 1-1.1 or 1-1.05 and after conjugation to a carrier protein, thepolydispersity of the conjugate is 1.0-2.5, 1.0-2.0. 1.0-1.5, 1.0-1.2,1.5-2.5, 1.7-2.2 or 1.5-2.0. All polydispersity measurements are byMALLS.

Saccharides are optionally sized up to 1.5, 2, 4, 6, 8, 10, 12, 14, 16,18 or 20 times from the size of the polysaccharide isolated frombacteria.

In one embodiment each N. meningitidis saccharide is either a nativepolysaccharide or is sized by a factor of no more than ×10. In a furtherembodiment each N. meningitidis capsular saccharide is a nativepolysaccharide. In a further embodiment at least one, two, three or fourN. meningitidis capsular saccharide(s) is sized by microfluidization. Ina further embodiment each N. meningitidis capsular saccharide is sizedby a factor of no more than ×10. In a further embodiment the N.meningitidis conjugates are made from a mixture of nativepolysaccharides and saccharides that are sized by a factor of no morethan ×10. In a further embodiment the capsular saccharide from serogroupY is sized by a factor of no more than ×10. In a further embodimentcapsular saccharides from serogroups A and C are native polysaccharidesand saccharides from serogroups W135 and Y are sized by a factor of nomore than ×10. In a further embodiment the average size of each N.meningitidis capular saccharide is between 50 kDa and 300 KDa or 50 kDaand 200 kDa. In a further embodiment the immunogenic compositioncomprises a MenA capsular saccharide having an average size of above 50kDa, 75 kDa, 100 kDa or an average size of between 50-100 kDa or 55-90KDa or 60-80 kDa. In a further embodiment the immunogenic compositioncomprises a MenC capsular saccharide having an average size of above 50kDa, 75 kDa, 100 kDa or between 100-200 kDa, 100-150 kDa, 80-120 kDa,90-110 kDa, 150-200 kDa, 120-240 kDa, 140-220 kDa, 160-200 kDa or190-200 kDa. In a further embodiment the immunogenic compositioncomprises a MenY capsular saccharide, having an average size of above 50kDa, 75 kDa, 100 kDa or between 60-190 kDa or 70-180 kDa or 80-170 kDaor 90-160 kDa or 100-150 kDa, 110-145 kDa or 120-140 kDa. In a furtherembodiment the immunogenic composition comprises a MenW capsularsaccharide having an average size of above 50 kDa, 75 kDa, 100 kDa orbetween 60-190 kDa or 70-180 kDa or 80-170 kDa or 90-160 kDa or 100-150kDa, 140-180 kDa, 150-170 kDa or 110-140 kDa.

The immunogenic composition of the invention may comprise a H.influenzae b capsular saccharide (Hib) conjugated to a carrier protein.This may be conjugated to a carrier protein selected from the groupconsisting of TT, DT, CRM197, fragment C of TT and protein D, forinstance TT. The Hib saccharide may be conjugated to the same carrierprotein as for at least one, two, three or all of the N. meningitidiscapsular saccharide conjugates, for instance TT. The ratio of Hib tocarrier protein in the Hib capsular saccharide conjugate may be between1:5 and 5:1 (w/w), for instance between 1:1 and 1:4, 1:2 and 1:3.5 oraround 1:3 (w/w). The Hib capsular saccharide may be conjugated to thecarrier protein via a linker (see above). The linker may bifunctional(with two reactive amino groups, such as ADH, or two reactive carboxylicacid groups, or a reactive amino group at one end and a reactivecarboxylic acid group at the other end). It may have between 4 and 12carbon atoms. Hib saccharide may be conjugated to the carrier protein orlinker using CNBr or CDAP. The carrier protein may be conjugated to theHib saccharide via the linker using a method comprising carbodiimidechemistry, for example EDAC chemistry (thus using the carboxyl chemicalgroup on the carrier). The dose of the Hib saccharide conjugate may bebetween 0.1 and 9 μg, 1 and 5 μg or 2 and 3 μg of saccharide.

In a further embodiment, the immunogenic composition of the inventioncomprises a Hib saccharide conjugate and at least two N. meningitidissaccharide conjugates wherein the Hib conjugate is present in a lowersaccharide dose than the mean saccharide dose of the at least two N.meningitidis saccharide conjugates. Alternatively, the Hib conjugate ispresent in a lower saccharide dose than the saccharide dose of each ofthe at least two N. meningitidis saccharide conjugates. For example, thedose of the Hib conjugate may be at least 10%, 20%, 30%, 40%, 50%, 60%,70% or 80% lower than the mean or lowest saccharide dose of the at leasttwo further N. meningitidis saccharide conjugates.

The mean dose is determined by adding the doses of all the furthersaccharides and dividing by the number of further saccharides. Furthersaccharides are all the saccharides within the immunogenic compositionapart from Hib and can include N. meningitidis capsular saccharides. The“dose” is in the amount of immunogenic composition or vaccine that isadministered to a human.

A Hib saccharide is the polyribosyl phosphate (PRP) capsularpolysaccharide of Haemophilus influenzae type b or an oligosaccharidederived therefrom.

At least two further bacterial saccharide conjugates is to be taken tomean two further bacterial saccharide conjugates in addition to a Hibconjugate. The two further bacterial conjugates may include N.meningitidis capular saccharide conjugates.

The immunogenic compositions of the invention may comprise furthersaccharide conjugates derived from one or more of Neisseriameningitidis, Streptococcus pneumoniae, Group A Streptococci, Group BStreptococci, S. typhi, Staphylococcus aureus or Staphylococcusepidermidis. In an embodiment, the immunogenic composition comprisescapsular saccharides derived from one or more of serogroups A, C, W135and Y of Neisseria meningitidis. A further embodiment comprises capsularsaccharides derived from Streptococcus pneumoniae. The pneumococcalcapsular saccharide antigens are optionally selected from serotypes 1,2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C,19A, 19F, 20, 22F, 23F and 33F (optionally from serotypes 1, 3, 4, 5,6B, 7F, 9V, 14, 18C, 19F and 23F). A further embodiment comprises theType 5, Type 8 or 336 capsular saccharides of Staphylococcus aureus. Afurther embodiment comprises the Type I, Type II or Type III capsularsaccharides of Staphylococcus epidermidis. A further embodimentcomprises the Vi saccharide from S. typhi. A further embodimentcomprises the Type Ia, Type Ic, Type II, Type III or Type V capsularsaccharides of Group B streptocoocus. A further embodiment comprises thecapsular saccharides of Group A streptococcus, optionally furthercomprising at least one M protein and optionally multiple types of Mprotein.

The immunogenic compositions of the invention may also comprise a DTPaor DTPw vaccine (for instance one containing DT, TT, and either a wholecell pertussis (Pw) vaccine or an acellular pertussis (Pa) vaccine(comprising for instance pertussis toxoid, FHA, pertactin, and,optionally agglutinogins 2 and 3). Such combinations may also comprise avaccine against hepatitis B (for instance it may comprise hepatitis Bsurface antigen [HepB], optionally adsorbed onto aluminium phosphate).In one embodiment the immunogenic composition of the invention comprisesa DTPwHepBHibMenAC vaccine where the HibMenAC component is as describedabove.

Immunogenic compositions of the invention optionally comprise additionalviral antigens conferring protection against disease caused by measlesand/or mumps and/or rubella and/or varicella. For example, immunogeniccomposition of the invention contains antigens from measles, mumps andrubella (MMR) or measles, mumps, rubella and varicella (MMRV). In anembodiment, these viral antigens are optionally present in the samecontainer as the meningococcal and/or Hib saccharide conjugate(s). In anembodiment, these viral antigens are lyophilised.

In an embodiment, the immunogenic composition of the invention furthercomprises an antigen from N. meningitidis serogroup B. The antigen isoptionally a capsular polysaccharide from N. meningitidis serogroup B(MenB) or a sized polysaccharide or oligosaccharide derived therefrom,which may be conjugated to a protein carrier. The antigen is optionallyan outer membrane vesicle preparation from N. meningitidis serogroup Bas described in EP301992, WO 01/09350, WO 04/14417, WO 04/14418 and WO04/14419.

In general, the immunogenic composition of the invention may comprise adose of each saccharide conjugate between 0.1 and 20 μg, 2 and 10 μg, 2and 6 μg or 4 and 7 μg of saccharide.

In an embodiment, the immunogenic composition of the invention containseach N. meningitidis capsular saccharide at a dose of between 0.1-20 μg;1-10 μg; 2-10 μg, 2.5-5 μg, around or exactly 5 μg; or around or exactly2.5 μg. In an embodiment, the immunogenic composition of the inventioncomprises MenA, MenC, MenW and MenY (optionally conjugated to tetanustoxoid) in doses of 2.5, 2.5, 2.5 and 2.5 μg respectively, 5, 5, 5 and 5μg respectively or 5, 5, 2, 5 and 2.5 μg respectively.

In an embodiment, the immunogenic composition of the invention forexample contains the Hib saccharide conjugate at a saccharide dosebetween 0.1 and 9 μg; 1 and 5 μg or 2 and 3 μg or around or exactly 2.5μg. In a further embodiment the immunogenic composition of the inventionfor example contains the Hib saccharide conjugate at a saccharide dosebetween 0.1 and 9 μg; 1 and 5 μg or 2 and 3 μg or around or exactly 2.5μg and each of the N. meningitidis polysaccharide conjugates at asaccharide dose of between 2 and 20 μg, 3 and 10 μg, or between 4 and 7μg or around or exactly 5 μg.

“Around” or “approximately” are defined as within 10% more or less ofthe given figure for the purposes of the invention.

In an embodiment, the immunogenic composition of the invention maycontain a saccharide dose of the Hib saccharide conjugate which is forexample less than 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% or 10% ofthe mean saccharide dose of at least two, three, four or each of the N.meningitidis saccharide conjugates. The saccharide dose of the Hibsaccharide is for example between 20% and 60%, 30% and 60%, 40% and 60%or around or exactly 50% of the mean saccharide dose of at least two,three, four or each of the N. meningitidis saccharide conjugates.

In an embodiment, the immunogenic composition of the invention containsa saccharide dose of the Hib saccharide conjugate which is for exampleless than 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of thelowest saccharide dose of the at least two, three, four or each of theN. meningitidis saccharide conjugates. The saccharide dose of the Hibsaccharide is for example between 20% and 60%, 30% and 60%, 40% and 60%or around or exactly 50% of the lowest saccharide dose of the at leasttwo, three, four or each of the N. meningitidis saccharide conjugates.

In an embodiment of the invention, the saccharide dose of each of the atleast two, three, four or each of the N. meningitidis saccharideconjugates is optionally the same, or approximately the same.

Examples of immunogenic compositions of the invention are compositionsconsisting of or comprising:

Hib conjugate and MenA conjugate and MenC conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:6:3, 1:3:3, 1:4:4,1:5:5, 1:6:6 (w/w). Optionally, the saccharide dose of MenA is greaterthan the saccharide dose of MenC.

Hib conjugate and MenC conjugate and MenY conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8:4, 1:6:3,1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w).

Optionally, the saccharide dose of MenC is greater than the saccharidedose of MenY.

Hib conjugate and MenC conjugate and MenW conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8:4, 1:6:3,1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally the saccharide dose of MenCis greater than the saccharide dose of MenW.

Hib conjugate and MenA conjugate and MenW conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8:4, 1:6:3,1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally, the saccharide dose ofMenA is greater than the saccharide dose of MenW.

Hib conjugate and MenA conjugate and MenY conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1, 1:8:4, 1:6:3,1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally the saccharide dose of MenAis greater than the saccharide dose of MenY.

Hib conjugate and MenW conjugate and MenY conjugate, optionally atsaccharide dose ratios of 1:2:2, 1:2:1, 1:1:2, 1:4:2, 1:2:4, 1:4:1,1:1:4, 1:3:6, 1:1:3, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionallythe saccharide dose of MenY is greater than the saccharide dose of MenW.

MenA, MenC, MenW and MenY at saccharide dose ratios of 1:1:1:1 or2:1:1:1 or 1:2:1:1 or 2:2:1:1 or 1:3:1:1 or 1:4:1:1 (w/w).

A further aspect of the invention is a vaccine comprising theimmunogenic composition of the invention and a pharmaceuticallyacceptable excipient.

In an embodiment, the immunogenic composition of the invention isadjusted to or buffered at, or adjusted to between pH 7.0 and 8.0, pH7.2 and 7.6 or around or exactly pH 7.4.

The immunogenic composition or vaccines of the invention are optionallylyophilised in the presence of a stabilising agent for example a polyolsuch as sucrose or trehalose.

Optionally, the immunogenic composition or vaccine of the inventioncontains an amount of an adjuvant sufficient to enhance the immuneresponse to the immunogen. Suitable adjuvants include, but are notlimited to, aluminium salts (aluminium phosphate or aluminiumhydroxide), squalene mixtures (SAF-1), muramyl peptide, saponinderivatives, mycobacterium cell wall preparations, monophosphoryl lipidA, mycolic acid derivatives, non-ionic block copolymer surfactants, QuilA, cholera toxin B subunit, polyphosphazene and derivatives, andimmunostimulating complexes (ISCOMs) such as those described byTakahashi et al. (1990) Nature 344:873-875.

For the N. meningitidis or HibMen combinations discussed above, it maybe advantageous not to use any aluminium salt adjuvant or any adjuvantat all.

As with all immunogenic compositions or vaccines, the immunologicallyeffective amounts of the immunogens must be determined empirically.Factors to be considered include the immunogenicity, whether or not theimmunogen will be complexed with or covalently attached to an adjuvantor carrier protein or other carrier, route of administrations and thenumber of immunising dosages to be administered.

The active agent can be present in varying concentrations in thepharmaceutical composition or vaccine of the invention. Typically, theminimum concentration of the substance is an amount necessary to achieveits intended use, while the maximum concentration is the maximum amountthat will remain in solution or homogeneously suspended within theinitial mixture. For instance, the minimum amount of a therapeutic agentis optionally one which will provide a single therapeutically effectivedosage. For bioactive substances, the minimum concentration is an amountnecessary for bioactivity upon reconstitution and the maximumconcentration is at the point at which a homogeneous suspension cannotbe maintained. In the case of single-dosed units, the amount is that ofa single therapeutic application. Generally, it is expected that eachdose will comprise 1-100 μg of protein antigen, optionally 5-50 μg or5-25 μg. For example, doses of bacterial saccharides are 10-20 μg, 5-10μg, 2.5-5 μg or 1-2.5 μg of saccharide in the conjugate.

The vaccine preparations of the present invention may be used to protector treat a mammal (for example a human patient) susceptible toinfection, by means of administering said vaccine via systemic ormucosal route. A human patient is optionally an infant (under 12months), a toddler (12-24, 12-16 or 12-14 months), a child (2-10, 3-8 or3-5 years) an adolescent (12-21, 14-20 or 15-19 years) or an adult.These administrations may include injection via the intramuscular,intraperitoneal, intradermal or subcutaneous routes; or via mucosaladministration to the oral/alimentary, respiratory, genitourinarytracts. Intranasal administration of vaccines for the treatment ofpneumonia or otitis media is preferred (as nasopharyngeal carriage ofpneumococci can be more effectively prevented, thus attenuatinginfection at its earliest stage). Although the vaccine of the inventionmay be administered as a single dose, components thereof may also beco-administered together at the same time or at different times (forinstance if saccharides are present in a vaccine these could beadministered separately at the same time or 1-2 weeks after theadministration of a bacterial protein vaccine for optimal coordinationof the immune responses with respect to each other). In addition to asingle route of administration, 2 different routes of administration maybe used. For example, viral antigens may be administered ID(intradermal), whilst bacterial proteins may be administered IM(intramuscular) or IN (intranasal). If saccharides are present, they maybe administered IM (or ID) and bacterial proteins may be administered IN(or ID). In addition, the vaccines of the invention may be administeredIM for priming doses and IN for booster doses.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenum Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

A further aspect of the invention is a vaccine kit for concomitant orsequential administration comprising two multi-valent immunogeniccompositions for conferring protection in a host against disease causedby Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriaeand Neisseria meningitidis and optionally Haemophilus influenzae. Forexample, the kit optionally comprises a first container comprising oneor more of:

tetanus toxoid (TT),

diphtheria toxoid (DT), and

whole cell or acellular pertussis components

and a second container comprising:

an immunogenic composition of the invention as described above (forinstance those comprising Men or HibMen saccharide conjugatecombinations).

A further aspect of the invention is a vaccine kit for concomitant orsequential administration comprising two multi-valent immunogeniccompositions for conferring protection in a host against disease causedby Streptococcus pneumoniae and Neisseria meningitidis and optionallyHaemophilus influenzae. For example, the kit optionally comprises afirst container comprising:

one or more conjugates of a carrier protein and a capsular saccharidefrom Streptococcus pneumoniae [where the capsular saccharide isoptionally from a pneumococcal serotype selected from the groupconsisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F].

and a second container comprising:

an immunogenic composition of the invention as described above (forinstance those comprising Men or HibMen saccharide conjugatecombinations).

Examples of the Hib conjugate and the N. meningitidis polysaccharideconjugates are as described above.

Typically the Streptococcus pneumoniae vaccine in the vaccine kit of thepresent invention (or in any of the immunogenic compositions of theinvention described above) will comprise saccharide antigens (optionallyconjugated), wherein the saccharides are derived from at least fourserotypes of pneumococcus chosen from the group consisting of 1, 2, 3,4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F,20, 22F, 23F and 33F. Optionally, the four serotypes include 6B, 14, 19Fand 23F. Optionally, at least 7 serotypes are included in thecomposition, for example those derived from serotypes 4, 6B, 9V, 14,18C, 19F, and 23F. Optionally more than 7 serotypes are included in thecomposition, for instance at least 10, 11, 12, 13 or 14 serotypes. Forexample the composition in one embodiment includes 10 or 11 capsularsaccharides derived from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and23F, and optionally 3 (all optionally conjugated). In an embodiment ofthe invention at least 13 saccharide antigens (optionally conjugated)are included, although further saccharide antigens, for example 23valent (such as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are alsocontemplated by the invention.

The pneumococcal saccharides are independently conjugated to any knowncarrier protein, for example CRM197, tetanus toxoid, diphtheria toxoid,protein D or any other carrier proteins as mentioned above.

Optionally, the vaccine kits of the invention comprise a thirdcomponent. For example, the kit optionally comprises a first containercomprising one or more of:

tetanus toxoid (TT),

diphtheria toxoid (DT), and

whole cell or acellular pertussis components

and a second container comprising:

one or more conjugates of a carrier protein and a capsular saccharidefrom Streptococcus pneumoniae [where the capsular saccharide isoptionably from a pneumococcal serotype selected from the groupconsisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F].

and a third container comprising:

an immunogenic composition of the invention as described above (forinstance those comprising Men or HibMen saccharide conjugatecombinations).

A further aspect of the invention is a process for making theimmunogenic composition or vaccine of the invention, comprising the stepof mixing the saccharides of the invention, for instance mixing N.meningitidis capsular saccharides from at least one, two, three or allfour of serogroups A, C, W and Y conjugated to a carrier protein with apharmaceutically acceptable excipient.

A further aspect of the invention is a method of immunising a human hostagainst disease caused by bacteria, for example N. meningitidis andoptionally Haemophilus influenzae infection comprising administering tothe host an immunoprotective dose of the immunogenic composition orvaccine or kit of the invention, optionally using a single dose.

An independent aspect of the invention is a method of immunising a humanhost with an immunogenic composition comprising at least 2 different N.meningitidis capsular saccharide conjugates selected from the groupconsisting of serogroup A, C, W and Y (optionally MenA, C, W and Y)wherein a single dose administration (optionally to teenagers, adults orchildren) results in a blood test taken one month after administrationgiving over 50%, 60%, 70%, 80%, 90% or 95% responders in an SBA assaymeasuring levels of response against MenA, MenC, MenW and/or MenY.Optionally the SBA assay is as described in Example 9 with responderassessed as described in Example 9.

A further independent aspect of the invention is an immunogeniccomposition comprising MenA, MenC, MenW and/or MenY conjugates which iscapable of eliciting an immune response after a single dose such thatover 50%, 60%, 70%, 80%, 90% or 95% of human subjects (children,teenagers or adults) inoculated are classified as responders in an SBAassay on blood extracted a month after inoculation (optionally using thecriteria described in example 9).

Such an immunogenic composition optionally has the further structuralcharacteristics described herein.

A further aspect of the invention is an immunogenic composition of theinvention for use in the treatment or prevention of disease caused bybacteria, for example N. meningitidis and optionally Haemophilusinfluenzae infection.

A further aspect of the invention is use of the immunogenic compositionor vaccine or kit of the invention in the manufacture of a medicamentfor the treatment or prevention of diseases caused by bacteria forexample N. meningitidis and optionally Haemophilus influenzae infection.

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting of”, “consist of” and “consists of”, respectively, in everyinstance.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

The invention is illustrated in the accompanying examples. The examplesbelow are carried out using standard techniques, which are well knownand routine to those of skill in the art, except where otherwisedescribed in detail. The examples are illustrative, but do not limit theinvention.

EXAMPLES Example 1—Preparation of Polysaccharide Conjugates

The covalent binding of Haemophilus influenzae (Hib) PRP polysaccharideto TT was carried out by a coupling chemistry developed by Chu et al.(Infection and Immunity 1983, 40 (1); 245-256). Hib PRP polysaccharidewas activated by adding CNBr and incubating at pH10.5 for 6 minutes. ThepH was lowered to pH8.75 and adipic acid dihydrazide (ADH) was added andincubation continued for a further 90 minutes. The activated PRP wascoupled to purified tetanus toxoid via carbodiimide condensation using1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDAC). EDAC was added tothe activated PRP to reach a final ratio of 0.6 mg EDAC/mg activatedPRP. The pH was adjusted to 5.0 and purified tetanus toxoid was added toreach 2 mg TT/mg activated PRP. The resulting solution was left forthree days with mild stirring. After filtration through a 0.45 μmmembrane, the conjugate was purified on a Sephacryl® S500HR (Pharmacia,Sweden) column equilibrated in 0.2M NaCl.

MenC-TT conjugates were produced using native polysaccharides (of over150 kDa as measured by MALLS) or were slightly microfluidised. MenA-TTconjugates were produced using either native polysaccharide or slightlymicrofluidised polysaccharide of over 60 kDa as measured by the MALLSmethod of example 2. MenW and MenY-TT conjugates were produced usingsized polysaccharides of around 100-200 kDa as measured by MALLS (seeexample 2). Sizing was by microfluidisation using a homogenizerEmulsiflex C-50 apparatus. The polysaccharides were then filteredthrough a 0.2 μm filter.

Activation and coupling were performed as described in WO96/29094 and WO00/56360. Briefly, the polysaccharide at a concentration of 10-20 mg/mlin 2M NaCl pH 5.5-6.0 was mixed with CDAPsolution (100 mg/ml freshlyprepared in acetonitrile/WFI, 50/50) to a final CDAP/polysaccharideratio of 0.75/1 or 1.5/1. After 1.5 minutes, the pH was raised withsodium hydroxide to pH10.0. After three minutes tetanus toxoid was addedto reach a protein/polysaccharide ratio of 1.5/1 for MenW, 1.2/1 forMenY, 1.5/1 for MenA or 1.5/1 for MenC. The reaction continued for oneto two hours.

After the coupling step, glycine was added to a final ratio ofglycine/PS (w/w) of 7.5/1 and the pH was adjusted to pH9.0. The mixturewas left for 30 minutes. The conjugate was clarified using a 10 μmKleenpak® filter and was then loaded onto a Sephacryl® S400HR columnusing an elution buffer of 150 mM NaCl, 10 mM or 5 mM Tris pH7.5.Clinical lots were filtered on an Opticap® 4 sterilizing membrane. Theresultant conjugates had an average polysaccharide:protein ratio of1:1-1:5 (w/w).

Example 1a—Preparation of MenA and MenC Polysaccharide Conjugates of theInvention

MenC-TT conjugates were produced using native polysaccharides (of over150 kDa as measured by MALLS) or were slightly microfluidised. MenA-TTconjugates were produced using either native polysaccharide or slightlymicrofluidised polysaccharide of over 60 kDa as measured by the MALLSmethod of example 2. Sizing was by microfluidisation using a homogenizerEmulsiflex C-50 apparatus. The polysaccharides were then filteredthrough a 0.2 μm filter.

In order to conjugate MenA capsular polysaccharide to tetanus toxoid viaa spacer, the following method was used. The covalent binding of thepolysaccharide and the spacer (ADH) is carried out by a couplingchemistry by which the polysaccharide is activated under controlledconditions by a cyanylating agent, 1-cyano-4-dimethylamino-pyridiniumtetrafluoroborate (CDAP). The spacer reacts with the cyanylated PSthrough its hydrazino groups, to form a stable isourea link between thespacer and the polysaccharide.

A 10 mg/ml solution of MenA (pH 6.0) [3.5 g] was treated with a freshlyprepared 100 mg/ml solution of CDAP in acetonitrile/water (50/50 (v/v))to obtain a CDAP/MenA ratio of 0.75 (w/w). After 1.5 minutes, the pH wasraised to pH 10.0. Three minutes later, ADH was added to obtain anADH/MenA ratio of 8.9. The pH of the solution was decreased to 8.75 andthe reaction proceeded for 2 hours maintaining this pH (with temperaturekept at 25° C.).

The PSAAH solution was concentrated to a quarter of its initial volumeand then diafiltered with 30 volumes of 0.2M NaCl using a Filtron Omegamembrane with a cut-off of 10 kDa, and the retentate was filtered.

Prior to the conjugation (carbodiimide condensation) reaction, thepurified TT solution and the PSAAH solution were diluted to reach aconcentration of 10 mg/ml for PSAAH and 10 mg/ml for TT.

EDAC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) was added to thePSAH solution (2 g saccharide) in order to reach a final ratio of 0.9 mgEDAC/mg PSAAH. The pH was adjusted to 5.0. The purified tetanus toxoidwas added with a peristaltic pump (in 60 minutes) to reach 2 mg TT/mgPSAAH. The resulting solution was left 60 min at +25° C. under stirringto obtain a final coupling time of 120 min. The solution was neutralisedby addition of 1M Tris-Hcl pH 7.5 ( 1/10 of the final volume) and left30 minutes at +25° C. then overnight at +2° C. to +8° C.

The conjugate was clarified using a 10 μm filter and was purified usinga Sephacryl® S400HR column (Pharmacia, Sweden). The column wasequilibrated in 10 mM Tris-HCl (pH 7.0), 0.075 M NaCl and the conjugate(approx. 660 mL) was loaded on the column (+2° C. to +8° C.). Theelution pool was selected as a function of optical density at 280 nm.Collection started when absorbance increased to 0.05. Harvest continueduntil the Kd reached 0.30. The conjugate was filter sterilised at +20°C., then stored at +2° C. to +8° C. The resultant conjugate had apolysaccharide:protein ratio of 1:2-1:4 (w/w).

In order to conjugate MenC capsular polysaccharide to tetanus toxoid viaa spacer, the following method was used. The covalent binding of thepolysaccharide and the spacer (ADH) is carried out by a couplingchemistry by which the polysaccharide is activated under controlledconditions by a cyanylating agent, 1-cyano-4-dimethylamino-pyridiniumtetrafluoroborate (CDAP). The spacer reacts with the cyanylated PSthrough its hydrazino groups, to form a stable isourea link between thespacer and the polysaccharide.

A 20 mg/ml solution of MenC (pH6.0) (3.5 g) was treated with a freshlyprepared 100 mg/ml solution of CDAP in acetonitrile/water (50/50 (v/v))to obtain a CDAP/MenC ratio of 1.5 (w/w). After 1.5 minutes, the pH wasraised to pH 10.0. At activation pH 5M NaCl was added to achieve a finalconcentration of 2M NaCl. Three minutes later, ADH was added to obtainan ADH/MenC ratio of 8.9. The pH of the solution was decreased to 8.75and the reaction proceeded for 2 hours (retained at 25° C.).

The PSCAH solution was concentrated to a minimum of 150 mL and thendiafiltered with 30 volumes of 0.2M NaCl using a Filtron Omega membranewith a cut-off of 10 kDa, and the retentate was filtered.

Prior to the conjugation reaction, the purified TT solution and thePSCAH solution (2 g scale) were diluted in 0.2M NaCl to reach aconcentration of 15 mg/ml for PSCAH and 20 mg/ml for TT.

The purified tetanus toxoid was added to the PSCAH solution in order toreach 2 mg TT/mg PSCAH. The pH was adjusted to 5.0. EDAC (16.7 mg/ml inTris 0.1M pH 7.5) was added with a peristaltic pump (in 10 minutes) toreach a final ratio of 0.5 mg EDAC/mg PSCAH. The resulting solution wasleft 110 min at +25° C. under stirring and pH regulation to obtain afinal coupling time of 120 min. The solution was then neutralized byaddition of 1M Tris-Hcl pH 9.0 ( 1/10 of final volume) and left 30minutes at +25° C. then overnight at +2° C. to +8° C.

The conjugate was clarified using a 10 μm filter and was purified usinga Sephacryl® S400HR column (Pharmacia, Sweden). The column wasequilibrated in 10 mM Tris-HCl (pH 7.0), 0.075 M NaCl and the conjugate(approx. 460 mL) was loaded on the column (+2° C. to +8° C.). Theelution pool was selected as a function of optical density at 280 nm.Collection started when absorbance increased to 0.05. Harvest continueduntil the Kd reached 0.20. The conjugate was filter sterilised at +20°C., then stored at +2° C. to +8° C. The resultant conjugate had apolysaccharide:protein ratio of 1:2-1:4 (w/w).

Example 2—Determination of Molecular Weight Using MALLS

Detectors were coupled to a HPLC size exclusion column from which thesamples were eluted. On one hand, the laser light scattering detectormeasured the light intensities scattered at 16 angles by themacromolecular solution and on the other hand, an interferometricrefractometer placed on-line allowed the determination of the quantityof sample eluted. From these intensities, the size and shape of themacromolecules in solution can be determined.

The mean molecular weight in weight (M_(w)) is defined as the sum of theweights of all the species multiplied by their respective molecularweight and divided by the sum of weights of all the species.

-   -   a) Weight-average molecular weight: -Mw-

$M_{w} = {\frac{\sum{W_{i} \cdot M_{i}}}{\sum W_{i}} = \frac{m_{2}}{m_{1}}}$

-   -   b) Number-average molecular weight: -Mn-

$M_{n} = {\frac{\sum{N_{i} \cdot M_{i}}}{\sum N_{i}} = \frac{m_{1}}{m_{0}}}$

-   -   c) Root mean square radius: -Rw- and R²w is the square radius        defined by:

${R^{2}w\mspace{14mu}{or}\mspace{14mu}\left( r^{2} \right)w} = \frac{\sum{m_{i} \cdot r_{i}^{2}}}{\sum m_{i}}$

-   -   -   (-m_(i)- is the mass of a scattering centre i and -r_(i)- is            the distance between the        -   scattering centre i and the center of gravity of the            macromolecule).

    -   d) The polydispersity is defined as the ratio -Mw/Mn-.

Meningococcal polysaccharides were analysed by MALLS by loading onto twoHPLC columns (TSKG6000 and 5000PWxl) used in combination. 25 μl of thepolysaccharide were loaded onto the column and was eluted with 0.75 mlof filtered water. The polyaccharides are detected using a lightscattering detector (Wyatt Dawn DSP equipped with a 10 mW argon laser at488 nm) and an inferometric refractometer (Wyatt Otilab DSP equippedwith a P100 cell and a red filter at 498 nm).

The molecular weight polydispersities and recoveries of all samples werecalculated by the Debye method using a polynomial fit order of 1 in theAstra 4.72 software.

Example 3—Clinical Trial Comparing Immunisation with Meningitec™ or aLarger Sized MenC-TT Conjugate

A phase II, open, controlled study was carried out to compareGSKBiologicals meningococcal serogroup C conjugate vaccine (MenC) with GSKBiological's Haemophilus influenzae b-meningococcal serogroup Cconjugate vaccine (Hib-MenC) or Meningitec™. Each dose of Meningitec™contains 10 μg of meningococcal serogroup C oligosaccharide conjugatedto 15 μg of CRM197 and is produced by Wyeth. The GSK MenC conjugatescontained native polysaccharides of about 200 kDa conjugated to tetanustoxoid (TT).

The study consisted of five groups, each planned to contain 100subjects, allocated to two parallel arms as follows:

In this present study, all subjects in both arms received one-fifth (⅕)of a dose of Mencevax™ ACWY and a concomitant dose of Infanrix™ hexa at12-15 months of age (Study Month 0). Two blood samples were collectedfrom all subjects (Study Month 0 and Study Month 1). Arm 1 consisted offour groups from a primary vaccination study who were primed at theirage of 3, 4 and 5 months with the following vaccines:

-   -   Group K: MenC (10 μg), non-adsorbed (non-ads), tetanus toxoid        (TT) conjugate and Infanrix™ hexa (MenC10-TT+Infanrix™ hexa)    -   Group L: Hib (10 μg)-MenC (10 μg), non-ads TT conjugate and        Infanrix™ penta (Hib10-MenC10-TT+Infanrix™ penta)    -   Group M: Hib (5 μg)-MenC (5 μg), non-ads, TT conjugate and        Infanrix™ penta (Hib5-MenC5-TT+Infanrix™ penta)    -   Group N: Meningitec™ and Infanrix™ hexa (Meningitec™+Infanrix™        hexa)

The two Hib-MenC-TT vaccine groups (Groups L and M) were kept blinded inthe booster study as to the exact formulation of the candidate vaccine.

Arm 2-(Group O) consisted of age-matched subjects not previouslyvaccinated with a meningococcal serogroup C vaccine (naïve) but who hadreceived routine pediatric vaccines according to the German PermanentCommission on Immunization.

Criteria for Evaluation:

Immunogenicity:

Determination of bactericidal antibody titers against meningococcal C(SBA-MenC) by a bactericidal test (cut-off: a dilution of 1:8) and ELISAmeasurement of antibodies against meningococcal serogroup C (assaycut-off: 0.3 μg/ml), the Hib polysaccharide PRP (assay cut-off: 0.15μg/ml) and tetanus toxoid (assay cut-off: 0.1 IU/ml) in blood samplesobtained prior to vaccination and approximately one month aftervaccination in all subjects.

Statistical Methods:

Demographics:

Determination of mean age in months (with median, range and standarddeviation [SD]), and racial and gender composition of the ATP and Totalvaccinated cohorts.

Immunogenicity:

Two analyses of immunogenicity were performed based on the ATP cohortfor immunogenicity (for analyses of immune memory and booster response)or the ATP cohort for safety (for analysis of persistence). Theseincluded: Evaluation of immune memory for MenC and booster response forHib and Tetanus (before and one month after administration of ⅕ dose ofthe plain polysaccharide vaccine):

-   -   Determination of geometric mean titers and concentrations (GMTs        and GMCs) with 95% confidence intervals (95% CI)    -   Determination of the percentage of subjects with antibody        titer/concentration above the proposed cutoffs with exact 95% CI        (seropositivity/seroprotection rates)    -   Investigation of antibody titers/concentration after vaccination        using reverse cumulative curves    -   Computation of standardized asymptotic 95% CI for the difference        in seropositivity/seroprotection rate    -   between the primed group (Groups K, L, M and N) and the unprimed        group (Group O)    -   Determination of the geometric mean of individual ratio of        SBA-MenC titer over anti-PSC concentration, with 95% CI    -   Determination of the 95% CI for the post-vaccination GMT/C ratio        between the groups K, L, M and the control group N for anti-PRP        and anti-tetanus and between each primed group (Groups K, L, M        and N) and the unprimed group (Group O) for SBA-MenC and        anti-PSC using an ANOVA model

Results

TABLE 1 SBA-MenC titres and anti-PSC antibody concentration afterbooster vaccination 95% CL 95% CL Antibody Group N GMT/C LL UL SBA-K-MenC-TT 71 3508.9 2580.1 4772.2 MenC L-HibMenC 79 2530.1 1831.7 3494.7M-HibMenC ™ 81 5385.4 4425.0 6554.2 N-Meningitec 85 1552.6 1044.4 2307.9O-Control 91 9.3 6.3 13.6 Anti- K-MenC-TT 70 28.10 22.59 34.95 PSCL-HibMenC 71 30.01 24.09 37.38 M-HibMenC ™ 76 34.58 29.10 41.09N-Meningitec 78 16.59 12.98 21.21 O-Control 94 3.05 2.36 3.93 Group K:subjects primed with MenC10-TT + Infanrix ™ hexa; Group L: subjectsprimed with Hib10-MenC10-TT + Infanrix ™ penta; Group M: subjects primedwith Hib5-MenC5-TT + Infanrix ™ penta; Group N: subjects primed withMeningitec ™ + Infanrix ™ hexa; Group O: control subjects (i.e. subjectsnot primed with MenC conjugate vaccine) N: number of subjects withavailable results

Higher titres of antibodies against MenC and higher SBA titres wereachieved by priming with the larger sized MenC polysaccharide conjugatevaccines (groups K, L and M) compared with the Meningitec™oligosaccharide conjugate vaccine.

TABLE 2 Geometric mean ratio for SBA MenC titre/anti-PSC concentrationGroup Timing N GMR LL UL K Pre 70 49.470 34.939 70.044 Post 66 126.138101.419 156.882 L Pre 76 36.528 25.849 51.621 Post 70 90.200 70.153115.975 M Pre 77 51.298 36.478 72.139 Post 74 164.950 139.304 195.318 NPre 84 22.571 16.521 30.837 Post 76 90.168 67.757 119.991 O Pre 3 91.6340.651 12889.8 Post 87 2.708 1.767 4.149

In all four primed groups (Groups K, L, M and N), the GMR increasedsignificantly from pre to post booster vaccination indicating thepresence of antibody maturation and functionality. GMR in the Group M(primed with Hib5-MenC5-TT) was higher than in the Group N (primed withMeningitec™)

TABLE 3 Persistence at 12-15 months of age just prior to administrationof the booster vaccines Differ- Value Endpoints Group N % Group N % ence% SBAMenC K 79 88.6 N 91 80.2 N − K −8.4 ≥1:8 L 84 93.3 N 91 80.2 N − L−3.1 M 85 87.1 N 91 80.2 N − M −6.8 SBAMenC K 79 65.8 N 91 51.6 N − K−14.2 ≥1:128 L 84 56.0 N 91 51.6 N − L −4.3 M 85 64.7 N 91 51.6 N − M−13.1 Anti-PSC K 79 100.0 N 91 100.0 N − K 0.0 ≥0.3 μg/ml L 84 100.0 N91 100.0 N − L 0.0 M 88 98.9 N 91 100.0 N − M 1.1 Anti-PSC K 79 72.2 N91 81.3 N − K 9.2 ≥2 μg/ml L 84 64.3 N 91 81.3 N − L 17.0 M 88 64.3 N 9181.3 N − M 8.6 Anti-PRP K 81 88.9 N 91 85.7 N − K −3.2 ≥0.15 μg/ml L 8696.5 N 91 85.7 N − L −10.8 M 90 98.9 N 91 85.7 N − M −13.2 Anti-PRP K 8133.3 N 91 28.6 N − K −4.8 ≥1 μg/ml L 86 55.8 N 91 28.6 N − L −27.2 M 9074.4 N 91 28.6 N − M −45.9 Anti- K 81 100.0 N 91 96.7 N − K −3.3 tetanusL 86 100.0 N 91 96.7 N − L −3.3 ≥0.1 IU/ml M 90 100.0 N 91 96.7 N − M−3.3 Group K: subjects primed with MenC10-TT + Infanrix ™ hexa; Group L:subjects primed with Hib10-MenC10-TT + Infanrix ™ penta; Group M:subjects primed with Hib5-MenC5-TT + Infanrix ™ penta; Group N: subjectsprimed with Meningitec ™ + Infanrix ™ hexa; N: number of subjects withavailable results Higher SBA titres against MenC were achieved bypriming with the larger size of MenC (groups K, L and M) compared topriming with the MenC-oligosaccharide conjugate Meningitec ™.

Immune Memory (ATP Cohort for Immunogenicity)

Administration of ⅕ dose of the plain polysaccharide ACWY vaccineelicited very high SBA-MenC titer in all four primed groups with98.7-100% and 97.5-100% of subjects primed with a candidate vaccineregimen exhibiting titers ≥1:8 and ≥1:128, respectively. In the groupprimed with the Meningitec™ regimen, there was a trend for a lowerpercentage of subjects with titers ≥1:128 (91.8%). In comparison, 17.6%of unprimed subjects had SBA MenC titers ≥1:8 and ≥1:128.

Example 4—Phase II Clinical Trial on HibMenAC-TT Conjugate Vaccine Mixedwith DTPw-HepB

Study Design:

Open, randomized (1:1:1:1:1), single centre study with five groups. Thefive groups received the following vaccination regimen respectively, at6, 10 and 14 weeks of age.

-   -   Tritanrix™-HepB/Hib-MenAC 2.5/2.5/2.5: henceforth referred to as        2.5/2.5/2.5    -   Tritanrix™-HepB/Hib-MenAC 2.5/5/5: henceforth referred to as        2.5/5/5    -   Tritanrix™-HepB/Hib-MenAC 5/5/5: henceforth referred to as 5/5/5    -   Tritanrix™-HepB+Hiberix™ henceforth referred to as Hiberix

Tritanrix™ HepB/Hiberix™+Meningitec™: henceforth referred to asMeningitec™

Blood samples were taken at the time of the first vaccine dose (Pre) andone month after the third vaccine dose (Post-dose 3).

Tritanrix™ is a DTPw vaccine marketed by GlaxoSmithKline BiologicalsS.A.

105 subjects were used in each of the five groups giving a total of 525subjects in the study.

TABLE 4 Content of GSK vaccine formulations Components per dose (0.5 ml)2.5/2.5/2.5* 2.5/5/5 5/5/5 Hib capsular polysaccharide PRP 2.5 μg 2.5μg   5 μg conjugated to tetanus toxoid (TT) Neisseria meningitidis Acapsular 2.5 μg 5 μg 5 μg polysaccharide (PSA) conjugated to TTNeisseria meningitidis C capsular 2.5 μg 5 μg 5 μg polysaccharide (PSC)conjugated to TT *The 2.5/2.5/2.5 vaccine was a dose dilution of GSKBiologicals' Hib-MenAC 5/5/5 vaccine containing 2.5 μg of each ofPRP-TT, MenA-TT and MenC-TT.

The Hib-MenAC vaccine formulations were mixed extemporaneously withTritanirix™-HepB. GSK Biologicals' combined diphtheria-tetanus-wholecell Bordetella pertussis-hepatitis B (DTPw-HB) vaccine(Tritanrix™-HepB) contains not less than 30 International Units (IU) ofdiphtheria toxoid, not less than 60 IU of tetanus toxoid, not less than41U of killed Bordetella pertussis and 10 μg of recombinant hepatitis Bsurface antigen.

Reference Therapy, Dose, Mode of Administration, Lot No.:

Vaccination Schedule/Site:

One group received Tritanrix™ HepB vaccine intramuscularly in the leftthigh and Hiberix™ intramuscularly in the right thigh at 6, 10 and 14weeks of age. Another group received Tritanrix™ HepB/Hiberix® vaccineintramuscularly in the left thigh and Meningitec™ vaccineintramuscularly in the right thigh at 6, 10 and 14 weeks of age.

Vaccine/Composition/Dose/Lot Number:

The Tritanrix™ HepB vaccine used was as described above.

One dose (0.5 ml) of GSK Biologicals' Haemophilus influenzae type bconjugate vaccine: Hiberix™ contained 10 μg of PRP conjugated to tetanustoxoid. In the Hiberix™ Group, it was mixed with sterile diluent and inthe Meningitec™ Group it was mixed with Tritanrix™-HepB.

One dose (0.5 ml) of Wyeth Lederle's MENINGITEC™ vaccine contained: 10μg of capsular oligosaccharide of meningococcal group C conjugated to 15μg of Corynebacterium diphtheria CRM197 protein and aluminium as salts.

Results—Immune Responses Generated Against Hib, MenA and MenC

TABLE 5a Anti-PRP (μg/ml) Group 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™Meningitec ™ % 95% CL % 95% CL 95% CL 95% CL 95% CL GMC/T LL UL GMC/T LLUL % GMC/T LL UL % GMC/T LL UL % GMC/T LL UL % ≥0.15 100 96.5 100 99.094.8 100 100 96.5 100 100 96.5 100 100 96.5 100 GMC 20.80 15.96 27.1022.62 17.72 28.88 19.36 15.33 24.46 38.55 29.93 49.64 10.94 8.62 13.88

TABLE 5b SBA-MenC Group 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™ Meningitec ™% 95% CL % 95% CL % 95% CL % 95% CL % 95% CL GMC/T LL UL GMC/T LL ULGMC/T LL UL GMC/T LL UL GMC/T LL UL % ≥1:8 99 94.7 100 100 96.5 100 10096.5 100 2.9 0.6 8.4 100 96.5 100 GMT 3132 2497 3930 4206 3409 5189 36973118 4384 4.7 3.9 5.6 4501 3904 5180

TABLE 5c SBA MenA Group 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™ Meningitec ™% 95% CL % 95% CL % 95% CL % 95% CL % 95% CL GMC/T LL UL GMC/T LL ULGMC/T LL UL GMC/T LL UL GMC/T LL UL % ≥1:8 99.7 91.9 99.7 100 95.8 100100 96.2 100 6.8 2.5 14.3 9.1 4.0 17.1 GMT 316.7 251.4 398.9 418.5 358.6488.5 363 310.5 424.4 5.6 4.3 7.4 5.6 4.4 7.2

TABLE 5d Anti-PSC (μg/ml) Group 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™Meningitec ™ % 95% CL % 95% CL % 95% CL % 95% CL % 95% CL GMC/T LL ULGMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL % ≥0.3 100 96.5 100 10096.4 100 100 96.5 100 8.2 3.6 15.6 100 96.5 100 GMC 49.03 43.24 55.5971.11 62.49 80.92 61.62 54.88 69.20 0.17 0.15 0.19 58.02 51.42 65.46

TABLE 5e Anti-PSA (μg/ml) Group 2.5/2.5/2.5 2.5/5/5 5/5/5 Hiberix ™Meningitec ™ % 95% CL % 95% CL % 95% CL % 95% CL % 95% CL GMC/T LL ULGMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL % ≥0.3 100 96.4 100 10096.5 100 99.0 94.8 100 1.0 0.0 5.4 5.9 2.2 12.5 GMC 18.10 15.34 21.3526.51 22.93 30.79 23.40 20.05 27.30 0.15 0.15 0.15 0.17 0.15 0.18

Conclusion

A comparison of the immunogenicity results achieved using theoligosaccharide MenC-CRM197 conjugate vaccine and the three GSKformulations which contain polysaccharide MenA-TT and MenC-TT conjugatesshowed that the polysaccharide Men conjugates were able to elicit a goodimmunogenic response similar to that achieved using the oligosaccharideconjugate vaccine Meningitec™. All formulations tested gave a responseto MenC in 100% of patients.

Example 5—Phase II Clinical Trial Administering Hib MenCY Concomitantlywith Infanrix™ Penta According to a 2, 3 and 4 Month Schedule

Study Design:

A Phase II, open (partially double-blind*) randomized controlledmulti-center study with 5 groups receiving a three-dose primary schedulewith vaccines as follows:

Group Hib-MenCY 2.5/5/5: Hib-MenCY (2.5/5/5)+Infanrix™ penta

Group Hib-MenCY 5/10/10: Hib-MenCY (5/10/10)+Infanrix™ penta

Group Hib-MenCY 5/5/5: Hib-MenCY (5/5/5)+Infanrix™ penta

Group Hib-MenC: Hib-MenC (5/5)+Infanrix™ penta

Group Menjugate: Menjugate™**+Infanrix™ hexa (control).

*Hib-MenCY 2.5/5/5, Hib-MenCY 5/10/10 and Hib-MenC were administered ina double-blind manner while the Hib-MenCY 5/5/5 group and the Menjugategroup were open. The 2.5/5/5, 5/10/10 and 5/5/5 formulations ofHib-MenCY contain MenC native polysaccharides and MenY polysaccharideswhich are microfluidized.

**Menjugate™ contains 10 μg of MenC oligosaccharides conjugated to12.5-25 μg of CRM197 per dose and is produced by Chiron.

Vaccination at +/−2, 3, 4 months of age (Study Month 0, Month 1 andMonth 2), and blood samples (3.5 ml) from all subjects prior to and onemonth post primary vaccination (Study Month 0 and Month 3).

Study Vaccine, Dose, Mode of Administration, Lot Number:

Three doses injected intramuscularly at one month intervals, atapproximately 2, 3 and 4 months of age as follows:

TABLE 6 Vaccines administered (study and control), group, schedule/siteand dose Concomitant Vaccine dose vaccine Schedule administeredadministered (months Site - Left upper Site Right upper Group of age)thigh thigh Hib-MenCY 2, 3, and 4 Hib (2.5 μg)- DTPa-HBV-IPV 2.5/5/5MenC-TT (5 μg)- (Infanrix ™ penta) MenY-TT (5 μg) Hib-MenCY 2, 3, and 4Hib (5 μg)-MenC- DTPa-HBV-IPV 5/10/10 TT (10 μg)-MenY- (Infanrix ™penta) TT (10 μg) Hib-MenCY 5/5/5 2, 3, and 4 Hib (5 μg)-MenC-DTPa-HBV-IPV TT (5 μg)-MenY- (Infanrix ™ penta) TT (5 μg) Hib-MenC 2, 3,and 4 Hib (5 μg)-Men C DTPa-HBV-IPV (5 μg) (Infanrix ™ penta)Menjugate ™ 2, 3, and 4 Menjugate ™ DTPa-HBV- IPV/Hib (Infanrix ™hexa)

Immunogenicity:

Measurement of antibody titres/concentrations against each vaccineantigen:

Prior to the first dose (Month 0) and approximately one month after thethird dose (Month 3) in all subjects for: SBA-MenC and SBA-MenY,anti-PSC and anti-PSY, anti-PRP, anti-T, anti-FHA, anti-PRN and anti-PT.Using serum bactericidal activity against N. meningitidis serogroups Cand Y (SBA-MenC and SBA-MenY cut-off: 1:8 and 1:128); ELISA assays withcut-offs: ≥0.3 μg/ml and ≥2 μg/ml for anti-N. meningitidis serogroups Cand Y polysaccharides (anti-PSC IgG and anti-PSY IgG); ≥0.15 μg/ml and≥1.0 μg/ml for Hib polysaccharide polyribosil-ribitol-phosphate(anti-PRP IgG); 5EL.U/ml for anti-FHA, anti-PRN, anti-PT; ≥0.1 IU/mlanti-tetanus toxoid (anti-TT). Only at one month after the third dose(Month 3) in all subjects for: anti-D, anti-HBs and anti-polio 1, 2 and3. Using ELISA assays with cut-offs: 0.1 IU/ml for anti-diphtheria(anti-D); 0 mIU/ml for antihepatitis B (anti-HBs); andmicroneutralization test cut-off: 1:8 for anti-polio type 1, 2 and 3(anti-polio 1, 2 and 3).

Statistical Methods:

The seroprotection/seropositivity rates and geometric meanconcentrations/titres (GMCs/GMTs) with 95% confidence intervals (95% CI)were computed per group, for SBA-MenC, anti-PSC, SBA-MenY, anti-PSY,anti-PRP, anti-Tetanus, anti-PT, anti-FHA and anti-PRN prior to and onemonth after vaccination; for anti-Diphtheria, anti-HBs, anti-Polio 1,anti-Polio 2 and anti-Polio 3 one month after vaccination. Vaccineresponse (appearance of antibodies in subjects initially seronegative orat least maintenance of antibody concentrations in subjects initiallyseropositive) with 95% CI for anti-PT, anti-PRN and anti-FHA were alsocomputed one month after vaccination. Reverse cumulative curves for eachantibody at Month 3 are also presented. The differences between theHib-MenCY and the Hib-MenC groups, compared with the Menjugate™ controlgroup were evaluated in an exploratory manner for each antibody, exceptfor SBA-MenY and anti-PSY, in terms of (1) the difference between theMenjugate™ group (minus) the Hib-MenCY and Hib-MenC groups for thepercentage of subjects above the specified cut-offs or with a vaccineresponse with their standardized asymptotic 95% CI, (2) the GMC or GMTratios of the Menjugate™ group over the Hib-MenCY and Hib-MenC groupswith their 95% CI. The same comparisons were done to evaluate thedifference between each pair of Hib-MenCY formulations for anti-PRP,SBA-MenC, anti-PSC, SBA-MenY, anti-PSY and anti-TT antibodies.

The overall incidences of local and general solicited symptoms werecomputed by group according to the type of symptom, their intensity andrelationship to vaccination (as percentages of subjects reportinggeneral, local, and any solicited symptoms within the 8 days followingvaccination and their exact 95% CI). Incidences of unsolicited symptomswere computed per group. For Grade 3 symptoms, onset 48 hours, medicalattention, duration, relationship to vaccination and outcomes wereprovided. Serious Adverse Events were fully described.

Seroprotection/Seropositivity Rates &GMC/Ts (ATP Cohort forImmunogenicity)

TABLE 7a Anti-PRP (μg/ml) Group N % ≥0.15 LL UL ≥1 LL UL GMC LL UL HibMenCY 2.5/5/5 67 100.0 94.6 100.0 98.5 92.0 100.0 9.01 7.25 11.21 HibMenCY 67 100.0 94.6 100.0 98.5 92.0 100.0 9.49 7.72 11.65 5/10/10 HibMenCY 5/5/5 70 100.0 94.9 100.0 98.6 92.3 100.0 8.08 6.53 9.98 Hib MenC74 100.0 95.1 100.0 98.6 92.7 100.0 10.44 8.49 12.83 Menjugate ™ 71100.0 94.9 100.0 80.3 69.1 88.8 2.60 1.97 3.43

TABLE 7b SBA-MenC (Titre) Group N % ≥1:8 LL UL ≥1:128 LL UL GMT LL ULHib MenCY 2.5/5/5 70 100.0 94.9 100.0 95.7 88.0 99.1 1005.8 773.5 1308.0Hib MenCY 67 100.0 94.6 100.0 94.0 85.4 98.3 1029.8 799.7 1326.0 5/10/10Hib MenCY 5/5/5 71 100.0 94.9 100.0 94.4 86.2 98.4 906.9 691.3 1189.8Hib MenC 74 100.0 95.1 100.0 95.9 88.6 99.2 871.0 677.3 1120.0Menjugate ™ 71 100.0 94.9 100.0 100.0 94.9 100.0 3557.6 2978.8 4248.8

TABLE 7c Anti-PSC (μg/ml) Group N % ≥0.3 LL UL ≥2 LL UL GMC LL UL HibMenCY 2.5/5/5 69 100.0 94.8 100.0 100.0 94.8 100.0 21.70 18.36 25.65 HibMenCY 66 100.0 94.6 100.0 100.0 94.6 100.0 27.26 23.26 31.95 5/10/10 HibMenCY 5/5/5 70 100.0 94.9 100.0 100.0 94.9 100.0 19.02 16.49 21.93 HibMenC 74 100.0 95.1 100.0 100.0 95.1 100.0 21.08 18.24 24.35 Menjugate ™71 100.0 94.9 100.0 100.0 94.9 100.0 38.49 33.64 44.05

TABLE 7d SBA-MenY (Titre) Group N % ≥1:8 LL UL ≥1:128 LL UL GMT LL ULHib MenCY 2.5/5/5 69 97.1 89.9 99.6 92.8 83.9 97.6 470.7 351.1 631.2 HibMenCY 66 97.0 89.5 99.6 86.4 75.7 93.6 437.1 322.0 593.4.8 5/10/10 HibMenCY 5/5/5 71 98.6 92.4 100.0 95.8 88.1 99.1 635.3 501.5 804.8 Hib MenC74 21.6 12.9 32.7 13.5 6.7 23.5 9.3 6.3 13.7 Menjugate ™ 71 19.7 11.230.9 9.9 4.1 19.3 7.5 5.4 10.4

TABLE 7e Anti-PSY (μg/ml) Group N % ≥0.3 LL UL ≥2 LL UL GMC LL UL HibMenCY 2.5/5/5 69 100.0 94.8 100.0 100.0 94.8 100.0 26.86 22.86 31.56 HibMenCY 66 100.0 94.6 100.0 100.0 94.6 100.0 37.02 31.84 43.04 5/10/10 HibMenCY 5/5/5 70 100.0 94.9 100.0 100.0 94.9 100.0 23.57 19.94 27.86 HibMenC 74 8.1 3.0 16.8 4.1 0.8 11.4 0.19 0.15 0.25 Menjugate ™ 71 5.6 1.613.8 1.4 0.0 7.6 0.17 0.15 0.19

TABLE 7f Anti-tetanus (IU/ml) Group N % ≥0.1 LL UL GMC LL UL Hib MenCY2.5/5/5 68 100.0 94.7 100.0 3.06 2.63 3.55 Hib MenCY 67 100.0 94.6 100.03.25 2.88 3.68 5/10/10 Hib MenCY 5/5/5 70 100.0 94.9 100.0 2.97 2.593.41 Hib MenC 74 100.0 95.1 100.0 3.15 2.73 3.64 Menjugate ™ 71 100.094.9 100.0 1.66 1.39 1.97 Group Hib-MenCY 2.5/5/5: Hib-MenCY (2.5/5/5) +Infanrix ™ penta Group Hib-MenCY 5/10/10: Hib-MenCY (5/10/10) +Infanrix ™ penta Group Hib-MenCY 5/5/5: Hib-MenCY (5/5/5) + Infanrix ™penta Group Hib-MenC: Hib-Men (5/5) + Infanrix ™ hexa Group Menjugate:Menjugate ™ + Infanrix ™ penta N = number of subjects with availableresults. % = percentage of subjects with concentration/titre within thespecificed range GMC/T: geometric mean concentration/titre 95% CI = 95%confidence interval; LL = Lower Limit; UL = Upper Limit

Conclusion

The MenC and Y polysaccharide conjugates produced a good immune responsein all subjects with 100% of subjects producing above 0.3 μg/mlresponses against MenC and MenY.

Example 6—Phase II Clinical Trial Comparing Three Formulations ofMenACWY-TT with Meningitec™ MenC-CRM197 Oligosaccharide-ConjugateVaccine

This example reports a phase II, open (partially-blind), randomized,controlled dose-range study to evaluate the Immunogenicity of threedifferent formulations of GlaxoSmithKline Biological's meningococcalserogroups A, C, W-135, Y tetanus toxoid conjugate (MenACWY-TT) vaccinein comparison to a MenC oligosaccharide-CRM197 conjugate vaccine(Meningitec™) when given as one dose to children aged 12-14 months.

The clinical trial was an open (partially double-blind*), controlled,multicentric study in which eligible subjects of 12-14 months wererandomized (1:1:1:1) to one of four parallel groups of 50 subjects toreceive a single primary dose at Visit 1 as follows:

Form 1T: MenACWY-TT at a dose of 2.5 μg of MenA polysaccharideconjugated to tetanus toxoid (TT), 2.5 μg of MenC polysaccharideconjugated to TT, 2.5 μg of MenW polysaccharide conjugated to TT and 2.5μg of MenY polysaccharide conjugated to TT.

Form 2T: MenACWY-TT at a dose of 5 μg of MenA polysaccharide conjugatedto TT, 5 μg of MenC polysaccharide conjugated to TT, 5 μg of MenWpolysaccharide conjugated to TT and 5 μg of MenY polysaccharideconjugated to TT.

Form 3T: MenACWY-TT at a dose of 2.5 μg of MenA polysaccharideconjugated to TT, 10 μg of MenC polysaccharide conjugated to TT, 2.5 μgof MenW polysaccharide conjugated to TT and 2.5 μg of MenYpolysaccharide conjugated to TT.

Ctrl T: 10 μg MenC oligosaccharide conjugated to 12.5-25 μg CRM197(Meningitec).

*The three different MenACWY-TT formulations were administered in adouble-blind manner.

Vaccination Schedule/Site:

A single vaccine dose was administered intramuscularly in the leftdeltoid at Visit 1 (Study Month 0) according to randomized assignment.All candidate vaccines were supplied as a lyophilized pellet in amonodose vial (0.5 ml after reconstitution with the supplied salinediluent).

Immunogenicity:

Measurement of titers/concentrations of antibodies against meningococcalvaccine antigen components in blood samples obtained prior to the studyvaccine dose (Month 0) and approximately one month after the studyvaccine dose (Month 1) in all subjects. Determination of bactericidalantibody titers against N. meningitidis serogroups A, C, W-135 and Y(SBA-MenA, SBA-MenC, SBA-MenW and SBA-MenY) by a bactericidal test(assay cut-offs: a dilution of 1:8 and 1:128) and ELISA measurement ofantibodies against N. meningitidis serogroups A, C, W-135 and Y(anti-PSA, anti-PSC, anti-PSW and anti-PSY, assay cut-offs ≥0.3 μg/mland ≥2 μg/ml), and tetanus toxoid (anti-tetanus, assay cut-off 0.1IU/ml).

Results

Antibody response in terms of the percentage of SBA-MenA, SBA-MenC,SBA-MenW and SBA-MenY responders one month after vaccination (theprimary endpoint) is shown in Table 8. A response is defined as greaterthan or equal to a 4-fold increase for seropositive subjects orseroconversion for seronegative subjects before vaccination.

TABLE 8 Vaccine responses for SBA antibody one month after vaccinationAntibody Group N % LL UL SBA-MenA Form 1T 42 61.9 45.6 76.4 Form 2T 3982.1 66.5 92.5 Form 3T 40 62.5 45.8 77.3 Meningitec ™ 36 11.1 3.1 26.1SBA-MenC Form 1T 46 97.8 88.5 99.9 Form 2T 43 100.0 91.8 100.0 Form 3T44 95.5 84.5 99.4 Meningitec ™ 49 91.8 80.4 97.7 SBA-MenW Form 1T 45100.0 92.1 100.0 Form 2T 43 97.7 87.7 99.9 Form 3T 45 100.0 92.1 100.0Meningitec ™ 46 15.2 6.3 28.9 SBA-MenY Form 1T 47 97.9 88.7 99.9 Form 2T44 88.6 75.4 96.2 Form 3T 45 93.3 81.7 98.6 Meningitec ™ 49 4.1 0.5 14.0

Table 9 shows the numbers of subjects achieving SBA titres over cutoffpoints of 1:8 and 1:128 as well as GMTs.

TABLE 9 Seropositivity rates and GMTs for SBA antibodies one month aftervaccination ≥1:8 ≥1:128 Group N % LL UL % LL UL GMT SBA-MenA Form 1T 46100 92.3 100 100 92.3 100 1457.3 Form2T 45 100 92.1 100 97.8 88.2 99.91776.9 Form3T 48 97.9 88.9 99.9 97.9 88.9 99.9 1339.5 Meningitec ™ 4151.2 35.1 67.1 43.9 28.5 60.3 42.8 SBA-MenC Form 1T 47 97.9 88.7 99.978.7 64.3 89.3 281.3 Form2T 45 100 92.1 100 84.4 70.5 93.5 428.6 Form3T47 95.7 85.5 99.5 85.1 71.7 93.8 478.4 Meningitec ™ 50 94.0 83.5 98.762.0 47.2 75.3 200.1 SBA-MenW Form 1T 47 100 92.5 100 100 92.5 1002529.1 Form2T 45 100 92.1 100 100 92.1 100 2501.6 Form3T 48 100 92.6 10097.9 88.9 99.9 2300.2 Meningitec ™ 48 27.1 15.3 41.8 6.3 1.3 17.2 9.4SBA-MenY Form 1T 47 100 92.5 100 100 92.5 100 1987.4 Form2T 45 100 92.1100 100 92.1 100 2464.8 Form3T 48 100 92.6 100 97.9 88.9 99.9 2033.7Meningitec ™ 49 49.0 34.4 63.7 28.6 16.6 43.3 25.0

Vaccination with all three formulations of the ACWY-TT polysaccharideconjugate led to good SBA responses against MenA, MenC, MenW and MenYwith 95-100% of subjects with titres greater than 1:8. In particular,the 5/5/5/5 and 2.5/10/2.5/2.5 formulations of the polysaccharideconjugates produced a higher response against MenC than theoligosaccharide Meningitec™ vaccine as seen by a higher proportion ofsubjects having a titre greater than 1:128 and the GMT readings.

TABLE 10 Seropositivity rates and GMCs for anti polysaccharideantibodies one month after vaccination ≥0.3 μg/ml ≥2 μg/ml GMC Group N %LL UL % LL UL μg/ml Anti- Form 1T 47 93.6 82.5 98.7 68.1 52.9 80.9 2.35MenA Form2T 45 100 92.1 100 64.4 48.8 78.1 3.11 Form3T 48 95.8 85.7 99.537.5 24.0 52.6 1.65 Meningitec ™ 50 10.0 3.3 21.8 2.0 0.1 10.6 0.18Anti- Form 1T 47 100 92.5 100 100 92.5 100 9.57 MenC Form2T 45 100 92.1100 100 92.1 100 12.53 Form3T 47 100 92.5 100 97.9 88.7 99.9 19.29Meningitec ™ 49 98.0 89.1 99.9 93.9 83.1 98.7 7.95 Anti- Form 1T 47 10092.5 100 80.9 66.7 90.9 4.56 MenW Form2T 45 100 92.1 100 93.3 81.7 98.66.83 Form3T 48 93.8 82.8 98.7 72.9 58.2 84.7 2.88 Meningitec ™ 50 0.00.0 7.1 0.0 0.0 7.1 0.15 Anti- Form 1T 47 100 92.5 100 97.9 88.7 99.98.90 MenY Form2T 45 100 92.1 100 100 92.1 100 12.78 Form3T 47 97.9 88.799.9 87.2 74.3 95.2 5.67 Meningitec ™ 50 2.0 0.1 10.6 0.0 0.0 7.1 0.15

All three formulations of the ACWY-TT polysaccharide conjugate vaccineproduced good immune responses against MenA, MenC, MenW and MenY withbetween 93% and 100% of subjects achieving titres grater than 0.3 μg/ml.Higher GMC readings were achieved using the 5/5/5/5 and 2/5/10/2.5/2.5formulations of the ACWY-TT polysaccharide conjugate vaccine incomparison with Meningitec™.

Example 7—Comparison of Immunogenicity of Native and Sized MenYPolysaccharide Conjugates

Mice (female DBA/2 of 6-8 wk) received two injections, 2 weeks apart, ofPSY-TT by the subcutaneous route. Blood samples were taken 14 days afterthe second injection in order to perform anti-PSY ELISA and SBA usingS1975 menY strain. Per injection, mice received 1 μg of PSY-TT (lyonon-ads formulation).

The conjugates described in table 11 were used.

TABLE 11 Conjugates ENYTT012 ENYTT014 ENYTT015 bis PSY NO Yes (40cycles) Yes (20 cycles) microfluidisation TT/PS ratio 1/1 1/1 1/1

Results

The results (FIG. 1) show a trend towards higher immunogenicity forconjugates prepared using sized PSY. FIG. 1A shows the GMC resultsobtained in an ELISA for antisera raised against conjugates preparedfrom native MenY (ENYTT012), microfluidised MenY—40 cycles (ENYTT014)and microfluidised MenY—20 cycles (ENYTT015 bis). Higher GMCs wereobtained where the MenY-TT was prepared from microfluidised MenY.

Similar results were obtained when the antisera were assessed by SBAassay (FIG. 1B). Again the higher GMT values were achieved usingconjugates prepared from microfluidised MenY.

Example 8—Clinical Trial Assessing the Effect of a Linker in MenA in aMenACWY Conjugate Vaccine

A single dose of different formulations of MenACWY vaccine wasadministered to teenagers of 15-19 years in 5 groups of 25 subjects in a1:1:1:1:1 randomised trial. The formulations tested were:

F1—MenACWY conjugated to tetanus toxoid with the MenA conjugatecontaining an AH spacer—5/5/5/5 μg

F2—MenACWY conjugated to tetanus toxoid with the MenA conjugatecontaining an AH spacer—2.5/5/2.5/2.5 μg

F3—MenACWY conjugated to tetanus toxoid with the MenA conjugatecontaining an AH spacer—5/5/2.5/2.5 μg

F4—MenACWY conjugated to tetansus toxoid with no spacer in anyconjugate—5/5/5/5 μg

Control group—Mencevax™ ACWY

On day 30 after inoculation, a blood sample was taken from the patients.

The blood samples were used to assess the percentage of SBA-MenA,SBA-MenC, SBA-MenW135 and SBA-MenY responders one month after thevaccine dose. A vaccine response was defined as 1) for initiallyseronegative subjects—a post-vaccination antibody titre 1/32 at 1 monthor 2) for initially seropositive subjects—antibody titre of 4 fold thepre-vaccination antibody titre.

Results

As shown in Table 12, the use of a spacer in the MenA conjugate led toan increased immune response against MenA. The percentage of respondersrose from 66% to 90-95% when the AH spacer was added. This was reflectedin an increase in SBA GMT from 4335 to 10000 and an increase in GMC from5 to 20-40. Surprisingly, the use of an AH spacer also led to anincreased immune response against MenC as seen by an increase in thepercentage of responders and an increase in the SBA GMT. An increasecould also be seen in the SBA-GMT against MenY (6742-7122) and againstMenW (4621-5418) when a spacer was introduced.

TABLE 12 % SBA MenA SBA-MenA Anti-PSA GMC Formulation responders GMTμg/ml ELISA F 1 5AH/5/5/5 90.9 9805 20.38 F2 2.5AH/5/2.5/2.5 75 851729.5 F3 5AH/5/2.5/2.5 95.5 10290  47.83 F4 5/5/5/5 66.7 4335 5.46Mencevax ™ 85.7 8022 27.39V % SBA MenC SBA-MenC Anti-PSC GMC Formulationresponders GMT μg/ml ELISA F 1 5AH/5/5/5 69.6 3989 12.11 F22.5AH/5/2.5/2.5 81.8 3524 12.78 F3 5AH/5/2.5/2.5 81.8 3608 8.4 F45/5/5/5 73.9 2391 8.84 Mencevax ™ 90.0 5447 38.71 % SBA MenW SBA-MenWAnti-PSW GMC Formulation responders GMT μg/ml ELISA F 1 5AH/5/5/5 955418 9.65 F2 2.5AH/5/2.5/2.5 85 4469 14.55 F3 5AH/5/2.5/2.5 95.5 42576.39 F4 5/5/5/5 95.5 4621 10.7 Mencevax ™ 86.4 2714 13.57 % SBY MenYSBA-MenY Anti-PSY GMC Formulation responders GMT μg/ml ELISA F 15AH/5/5/5 91.3 7122 16.3 F2 2.5AH/5/2.5/2.5 87.5 5755 12.52 F35AH/5/2.5/2.5 80 5928 8.88 F4 5/5/5/5 91.3 6742 13.88 Mencevax ™ 91.74854 21.02

Example 9—Clinical Trial Assessing the Effect of a Linker in MenA andMenC Conjugates in a MenACWY Conjugate Vaccine

A single dose of different formulations of MenACWY vaccine wasadministered to teenagers of 15-19 years in 5 groups of 25 subjects in a1:1:1:1:1 randomised trial. The formulations tested were:

F1—MenACWY conjugated to tetanus toxoid with the MenA and MenCconjugates containing an AH spacer—2.5/2.5/2.5/2.5 μg

F2—MenACWY conjugated to tetanus toxoid with the MenA and MenCconjugates containing an AH spacer—5/5/2.5/2.5 μg

F3—MenACWY conjugated to tetanus toxoid with the MenA and MenCconjugates containing an AH spacer—5/5/5/5 μg

F4—MenACWY conjugated to tetansus toxoid with the MenA conjugatecontaining an AH spacer—5/5/5/5 μg

Control group—Mencevax™ ACWY

On day 30 after inoculation, a blood sample was taken from the patients.

The blood samples were used to assess the percentage of SBA-MenA,SBA-MenC, SBA-MenW135 and SBA-MenY responders one month after thevaccine dose. A vaccine response was defined as 1) for initiallyseronegative subjects—a post-vaccination antibody titre ≥ 1/32 at 1month or 2) for initially seropositive subjects—antibody titre of ≥4fold the pre-vaccination antibody titre.

Results

The introduction of an AH spacer into the MenC conjugate led to anincrease in the immune response against MenC as shown in Table 13. Thisis demonstrated by an increase in SBA GMT from 1943 to 4329 and anincrease in anti-PSC GMC from 7.65 to 13.13. Good immune responsesagainst MenA, MenW and MenY were maintained.

TABLE 13 Anti-PSA % SBA MenA SBA-MenA GMC Formulation responders GMTμg/ml ELISA F 12.5AH/2.5AH/2.5/2.5 75 8417 20.23 F2 5AH/5AH/2.5/2.5 726299 16.07 F3 5AH/5AH/5/5 87 9264 27.26 F4 5AH/5/5/5 77.3 9632 20.39Mencevax ™ 78.3 8284 12.93 Anti-PSC % SBA MenC SBA-MenC GMC Formulationresponders GMT μg/ml ELISA F 12.5AH/2.5AH/2.5/2.5 88 3619 12.82 F25AH/5AH/2.5/2.5 88 2833 13.32 F3 5AH/5AH/5/5 95.8 4329 13.13 F45AH/5/5/5 95.8 1943 7.65 Mencevax ™ 91.7 1567 16.55 Anti-PSW % SBA MenWSBA-MenW GMC Formulation responders GMT μg/ml ELISA F12.5AH/2.5AH/2.5/2.5 100 5656 7 F2 5AH/5AH/2.5/2.5 96 4679 5.4 F35AH/5AH/5/5 91.3 4422 4.45 F45AH/5/5/5 88 4947 7.67 Mencevax ™ 96 348611.93 Anti-PSY % SBY MenY SBA-MenY GMC Formulation responders GMT μg/mlELISA F 1 75 3891 17.81 2.5AH/2.5AH/2.5/2.5 F2 5AH/5AH/2.5/2.5 92 396811.96 F3 5AH/5AH/5/5 79.2 2756 9.51 F4 5AH/5/5/5 80 3914 16.76Mencevax ™ 88 3056 21.41

The invention claimed is:
 1. An immunogenic composition, comprising: (a)a Neisseria meningitidis (N. meningitides) serogroup A capsularsaccharide conjugated to an adipic acid dihydrazide (ADH) linker,wherein the linker is conjugated to tetanus toxoid carrier protein; (b)a N. meningitidis serogroup C capsular saccharide conjugated to anadipic acid dihydrazide (ADH) linker, wherein the linker is conjugatedto tetanus toxoid carrier protein; (c) a N. meningitidis serogroup Wcapsular saccharide directly conjugated to tetanus toxoid carrierprotein in the absence of a linker; and (d) a N. meningitidis serogroupY capsular saccharide directly conjugated to tetanus toxoid carrierprotein in the absence of a linker; wherein the ratio of Men Asaccharide to carrier protein is between 1:2 to 1:5 (w/w); and whereinthe ratio of MenC saccharide to carrier protein is between 1:2 to 1:5(w/w).
 2. The immunogenic composition of claim 1, wherein the ratio ofMen W and/or Y saccharide to carrier protein is between 1:0.5 and 1:2(w/w).
 3. The immunogenic composition of claim 1, wherein thecomposition does not include an adjuvant.
 4. The immunogenic compositionof claim 1, wherein the composition does not include an aluminum saltadjuvant.
 5. The immunogenic composition of claim 1, further comprisingsucrose.
 6. The immunogenic composition of claim 1, further comprising aN. meningitidis serogroup B outer membrane vesicle preparation orcapsular saccharide.
 7. The immunogenic composition of claim 1, furthercomprising Haemophilus influenza (H. influenzae) b capsular saccharideconjugated to a carrier protein, the carrier protein selected from thegroup consisting of tetanus toxoid, diphtheria toxoid, cross reactivematerial 197, fragment C of tetanus toxoid and protein D, wherein the H.influenzae b conjugate is present in a lower dose than the dose of anyother bacterial saccharide conjugate.
 8. The immunogenic composition ofclaim 1, wherein the H. influenzae b capsular saccharide (Hib) isconjugated to tetanus toxoid.
 9. A pharmaceutical composition,comprising the immunogenic composition of claim 1 and a pharmaceuticallyacceptable excipient.
 10. A process for making a vaccine, comprising thestep of mixing the immunogenic composition of claim 1 with apharmaceutically acceptable excipient.
 11. A method of immunizing ahuman host against disease caused by Neisseria meningitidis infection,comprising administering to the host an immunoprotective dose of theimmunogenic composition of claim 1.